Plants having increased tolerance to heat stress

ABSTRACT

The invention relates to methods of producing a desired phenotype in a plant by manipulation of gene expression within the plant. The method relates to means which increase the level of expression of a MYB-subgroup14 polynucleotide or a MYB68 polypeptide. The method also relates to expression of a nucleic acid sequence encoding a MYB-subgroup14 or a MYB68 transcriptional factor. The methods are directed to elevating the levels of a MYB-subgroup14 or a MYB68 expression, wherein a desired phenotype such as reduced flower abortion and increased yield during heat stress is observed. The invention also relates to nucleic acid sequences useful in such methods.

REFERENCE TO RELATED APPLICATIONS

This non-provisional patent application is a continuation of pendingU.S. patent application Ser. No. 12/148,548, filed Apr. 18, 2008, whichclaims priority under 35 U.S.C. §119(e) to U.S. Ser. No. 60/925,312,filed Apr. 18, 2007, and U.S. Ser. No. 60/965,582, filed Aug. 20, 2007,the contents of each of which are herein incorporated by reference intheir entireties.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The contents of the text file named “PREP-014C01US SequenceListing.txt”, which was created on Jan. 26, 2017 and is 439 KB in size,are hereby incorporated by reference in their entireties.

FIELD OF THE INVENTION

The invention is in the field of plant molecular biology and relates totransgenic plants having novel phenotypes, methods of producing suchplants and polynucleotides and polypeptides useful in such methods. Morespecifically, the invention relates to the use of MYB polynucleotidesand transgenic plants expressing these polynucleotides and polypeptides.

BACKGROUND OF THE INVENTION

Environmental stresses are responsible for significant yield reductionin agricultural crops. In addition to many reports published previously,the relation between climate variation and production of corn andsoybean throughout the United States for the period 1982-1998 wasstudied in recent years (Lobell and Asner, 2003). Gradual temperaturechanges have made a measurable impact on crop yield. In corn and soybeanit has been estimated that yield is reduced by 17% per degree as thegrowth temperature rises above the season optimum. With a predictedtemperature increase of 1.4° C. to 5.8° C. between the years 1990 and2010 (IPCC Working Group I, 2001), improvement of high temperaturetolerance in crop plants has become one of the major focuses ofagricultural biotechnology development.

Both monocots and dicots are particularly sensitive to heat stressduring flowering and seed development and therefore heat stress has asignificant impact on seed yield (Young et al., 2004; Sato et al., 2002;Angadi et al., 2000; Carlson, 1990; Wahid, A., Gelani, S., Ashraf, M.,and Foolad, M. R. (2007)). It has been suggested that plants possess aninherent ability for basal and acquired thermotolerance and that acommon heat response mechanisms is present in diverse plant species(Kapoor et al., 1990; Vierling, 1991; Flahaut et al., 1996; Burke etal., 2000; Hong and Vieling, 2000; Massie et al., 2003; Larkindale etal., 2005). Basal thermotolerance allows plants survive from exposure totemperature above optimal for growth, whereas acquired thermotoleranceis induced by a short acclimation period at a sub-lethal heat stresswhich enables a plant to survive a subsequent heat stress that would beotherwise lethal. A number of studies have been conducted to identifyand characterize genes and pathways that are involved in plantthermotolerance. For example, heat shock transcription factors (HSF) andheat shock proteins (HSP) have received much attention to elucidate theroles and effects of these genes in response to heat stress as haveplant growth hormones such as abscisic acid and ethylene.

Transcription factors are DNA binding proteins that interact withspecific promoter or enhancer sequences and alter the gene expression ofthe associated gene. Where the specific sequence that binds thetranscription factor is associated with a suite of genes whole pathwayscan be coordinately regulated with various component genes beingsimultaneously up-regulated or down-regulated. A transcription factorsmay coordinately alter a suite of genes in response to a stimulus suchas an environmental stress, nutritional status or pathogen attack, forexample, or can be a component of a signaling pathway, such as a hormonesignaling pathway for example. Transcription factors posses a modularstructure and are classified primarily on the basis of the DNA bindingdomain.

The MYB family of transcription factors is composed of at least 198genes (Yanhui et al. 2006) and has been proposed to have regulatoryfunctions in a wide array of processes ranging from growth anddevelopment to defense responses. Plant MYB proteins are classifiedbased on the presence and number of imperfect MYB repeats each composedof about 52 amino acids. The MYB domain forms a helix-turn-helixconformation and represents the DNA binding domain. Three major groupsof MYB proteins have been classified as R1R2R3-MYB, R2R3-MYB andMYB-related proteins.

The R2R3-MYB family of proteins in Arabidopsis consists of 125 proteinsand is characterized by having a R2R3 DNA binding domain at theirN-terminus (Kranz et al., 1998, and Stracke et al., 2001). These genesare involved in a number of biological processes including mediatinghormone actions, secondary metabolism (Paz-Ares et al., 1987), controlof cell morphogenesis (Oppenheimer et al., 1991), meristem, floral andseed development (Kirik et al., 1998, Schmitz et al., 2002) and responseto various environmental factors (Kranz et al., 1998; Jin and Martin,1999; Meissner et al, 1999).

MYB sequences have been further classified into a number of subgroupsbased on sequence (Krantz et al 1998, Stracke et al 2001). MYB68 fallswithin subgroup14 as does MYB36 and MYB84 as identified by Krantz et al1998. However, Stracke et al 2001, have additionally include the MYB37,MYB38 and MYB87 in subgroup14. Stracke further notes that there areseveral cases of functional conservation of genes that cluster togetherin the dendrogram.

Classification of the R2R3-MYB family has identified 125 MYB proteins inArabidopsis thaliana (At). A R2R3 MYB gene is characterized by a MYBdomain containing two imperfect repeats of 53 aa (R2, and R3). Eachrepeat contains three helix-turn-helix structures. The R2 and R3 domainsare located near the N-terminus of the proteins. The last two helices oneach repeat with a loop between them form a DNA-binding motif structuresimilar to HLH proteins. The third helix directly binds to DNA, and thefirst and second helices contribute to the conformation of the HLH motifthat appears to be important in recognition of a specific gene target(Ogata et al., 1994; William and Grotewold, 1997; Jia et al., 2004). TheR2R3-MYB proteins were further characterized into 22 subgroups accordingto their phylogenetic relationship based on at least one of the sharedamino acid motifs in addition to the MYB domain (Kranz et al., 1998).AtMYB68, AtMYB84, and AtMYB36 were categorized as subgroup14 based ontwo shared motifs: S1: SFSQLLLDPN SEQ ID NO:266 and S2: TSTSADQSTISWEDISEQ ID NO:267, at the C-terminus of the proteins. The homology at thesemotifs was limited, for example, Arabidopsis MYB36 has only 20%identity. Subsequently, AtMYB87, AtMYB37 and AtMYB38 were also includedin subgroup14, on the basis of sequence conservation in the MYB DNAdomain: R2 and R3 helix-turn-helix repeats (Stracke et al., 2001).

The R2R3 domains may be indicative of specific DNA binding through theunique amino acid sequence of the third helix of the R3 domain and minorconformational changes associated with the structural interactionbetween the first two helices. It suggests that subgroup14 members maybe functionally redundant orthologous. For example, lateral meristeminitiation in Arabidopsis was studied with respect to MYB-subgroup14(Muller et al., 2006). All members of MYB-subgroup14 showed highsimilarity to the tomato Blind (B1) gene, a regulator of axillarymeristems. Transcripts of four members: AtMYB37, AtMYB38, AtMYB84 andAtMYB87 were detected by RT-PCR in tissues including shoot tip,internode, leaf, flower bud, open flower, and root, whereas AtMYB36 andAtMYB68 expression was expressed in root tissue. Phenotypic analysisusing knockouts of AtMYB37, AtMYB38 and AtMYB84 indicated that thesemembers of MYB-subgroup14 at least partially redundant for regulatingaxillary bud formation.

MYB68 is a R2R3 type MYB gene, and a member of MYB-subgroup14, that hasbeen identified in a transposon gene trapping study (Feng et al., 2004).Expression of this gene has been demonstrated to be specific to rootpericycle cells. In the null mutant, no MYB68 mRNA was detectable;however, no mutant phenotype was exhibited when plants were grown understandard conditions. In the evaluation of MYB68 under a variety ofgrowth conditions the only phenotype discerned was reduced plant leafarea when plants were grown under hot greenhouse conditions (30-40° C.).This phenotype was rescued by transformation of the myb68 mutantbackground with a wild-type MYB68 gene. Examination root tissue of themyb68 mutant grown in root cultures indicated increased biomass andlignin levels. The authors conclude that MYB68 is involved in rootdevelopment (Feng et al., 2004).

Transcriptional activation is primarily mediated through transcriptionfactors that interact with enhancer and promoter elements. Binding oftranscription factors to such DNA elements constitutes a crucial step intranscriptional initiation. Each transcription factor binds to itsspecific binding sequence in a promoter and activates expression of thelinked coding region through interactions with coactivators and/orproteins that are a part of the transcription complex.

SUMMARY OF THE INVENTION

This invention relates to a method for enhancing the heat stresstolerance of plants by means of increasing the expression of a MYBsubgroup-14 polypeptide. Enhanced heat stress tolerance includesimproved seed set during and following conditions of heat stress.Improved seed set results in increased yield. A MYB-subgroup-14polypeptide includes for example a MYB68, a MYB36, a MYB84, a MYB37,aMYB38 or a MYB87 polypeptide. Preferably, the MYB-subgroup-14polypeptide is a MYB68, a MYB36 or a MYB84 polypeptide. The MYBsubgroup-14 polypeptide expression is ectopic, or constitutive.Alternatively, expression of the MYB subgroup-14 polypeptide in itstypical place of expression, e.g. root tissue.

A heat stress heat stress tolerant plant is produced by providing anucleic acid construct that increases the expression of a Mybsubgroup-14 polypeptide, inserting the nucleic construct into a vector,transforming a plant, tissue culture, or a plant cell with the vector toobtain a plant, tissue culture or a plant cell with increased expressionof the Myb subgroup-14 polypeptide and growing said plant orregenerating a plant from the tissue culture or plant cell. A nucleicacid construct that increases the expression of a Myb subgroup-14polypeptide includes for example an enhancer element. An enhancer is asequence found in eukaryotes and certain eukaryotic viruses which canincrease transcription of a gene when located, in either orientation, upto several kilobases from the gene concerned. These sequences act asenhancers when on the 5′ side (upstream) of the gene in question.However, some enhancers are active when placed on the 3′ side(downstream) of the gene. The enhancer elements can activatetranscription of a gene and alter the normal expression pattern of theendogenous gene. Enhancer elements are known to those skilled in theart. For example the enhancer element is a 35S enhancer element.

Additionally, a nucleic acid construct that increases the expression ofa Myb subgroup-14 polypeptide includes for example a nucleic acidencoding a Myb subgroup-14 polypeptide. Exemplary, MYB polypeptides andnucleic acids include those of SEQ ID NO: 1-265. The nucleic acidencoding a Myb subgroup-14 polypeptide is operably linked to a promoter.The promoter is a heterologous promoter or a homologous promoter.Additionally, the promoter is a constitutive or an inducible promoter.

By increasing the expression of a MYB subgroup-14 polypeptide is meantthat the amount produced by the cell transformed with the nucleic acidconstruct is greater than a cell, e.g. control cell that is nottransformed with the nucleic acid construct. A control cell includes forexample a cell that endogenously expresses a MYB subgroup-14 polypeptidesuch a plant root cell, alternatively a control cell is a nontransformed cell of the same cell-type as the transformed cell, be it aleaf cell a meristem cell or a flower or seed cell. An increase is a1-fold, 2-fold, 3 fold or greater increase. An increase of expression isalso meant to include expression of a MYB subgroup-14 polypeptide in acell that does not typically produced by a cell.

Also included in the invention is a method of identifying a heat stresstolerant plant. The plants identified by these methods have reducedflower abortion and increased yield as compared to a control plant. Heatstress tolerant plants are identified by exposing a population offlowering plants to a heat stress treatment and selecting a plant fromthe population of plants that has reduced flower abortion. Heat stresstreatment includes for example exposing the plant to a temperature thatis hot enough for a sufficient amount of time such that damage to plantfunctions or development results. By reduced flower abortion is meantthat a plant does not loss as many flowers, due to flower abortion, orhas a greater seed yield compared to another plant that is exposed to asimilar level of heat stress. Plants with a reduced flower abortion havea 5, 10, 20, 25, 30% or more increase in seed yield as compared to acontrol plant.

The invention further includes the plants produced by the methods of theinvention and the seed produced by the plants which produce a plant thathas an increase tolerance to heat stress.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DETAILED DESCRIPTION

The invention is based upon the surprising discovery of plants that havean increased tolerance to heat stress which results in an increasedyield relative to a wild-type control. More specifically, the inventionis based upon the discovery that increasing the expression of aMYB-subgroup14 polypeptide (e.g., MYB68) results in a plant having anincreased resistance to heat stress.

Expression of a MYB-subgroup 14 polypeptide can be accomplished forexample by increasing the expression of an endogenous MYB-subgroup 14polypeptide (e.g., activation tag insertion) or by expression of anexogenous gene construct encoding for a MYB-subgroup 14 polypeptide. Thegene encoding for the MYB-subgroup 14 polypeptide may be endogenous orexogenous to the transformed species. As shown in the EXAMPLES plantshaving an increases resistance to heat stress were produced not onlytransforming a plant with its native MYB-subgroup 14 polypeptide butalso with a MYB-subgroup 14 polypeptide from another plant species.

Accordingly the invention provides methods of enhancing (e.g.,increasing) the heat stress tolerance of plants by increasing theexpression of a MYB subgroup-14 polypeptide. Also included in theinvention is a method of identifying a heat stress tolerant plant. Theplants identified by these methods have reduced flower abortion andincreased yield as compared to a control plant. Heat stress tolerantplants are identified by exposing the population of flowering plants toa heat stress treatment and selecting a plant from the population ofplants that has reduced flower abortion. The invention also includes thetransgenic plants produced by the methods of the invention and the seedsproduced by the transgenic plants that produce a heat stress tolerantplant.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In the case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

For convenience, before further description of the present invention,certain terms employed in the specification, examples and appendedclaims are defined herein. These definitions should be read in light ofthe remainder of the disclosure and as understood by a person ofordinary skill in the art.

The term “constitutive expression” means expression of a gene in anycell at constant levels in a non-regulated manner.

The terms “cMYB” and “MYB” refer to a cDNA clone of MYB and are usedinterchangeably. Where a genomic sequence has been used or referred to,it is identified and differentiated by the term “gMYB” or “genomic MYB”thereby referring to a genomic MYB sequence.

The term “ectopic expression” means expression of a gene in an abnormalplace in an organism relative to the endogenous gene expression. Ectopicexpression may include constitutively expressed genes depending on thenative expression patterns of a given gene.

The term “expression cassette” means a vector construct wherein a geneis transcribed. Additionally, the expressed mRNA may be translated intoa polypeptide.

The terms “expression” or “over-expression” are used interchangeably andmeans the expression of a gene such that the transgene is expressed. Thetotal level of expression in a cell may be elevated relative to awild-type cell.

“Flower abortion” means a flower that fails to develop and produce afruit or seed. In addition to premature senescence of a flower, flowerabortion may refer to loss of pollen production, altered pollination orfertilization and subsequent seed development. Altered growth anddevelopment of meristem tissue, a flower meristem in particular, isfurther included within the meaning of flower abortion.

The term “heat tolerance” is defined as a phenotype where a first plant,or plant line, has increased capacity to withstand elevated temperatureand produce a yield that is in excess of a second plant or plant line,the second plant line being a control plant such as a wild-type controlplant line.

A “promoter sequence”, or “promoter”, means a nucleic acid sequencecapable of inducing transcription of an operably linked gene sequence ina plant cell.

The term “seed set” is seed formation as a result of flower pollinationfollowed by egg cell fertilization and zygote development. Reductions inseed set which can occur due to interruption in any of the aboveprocesses will produce a net reduction in seed number produced.

The term “substantially similar” refers to nucleic acids where a changein one or more nucleotides does not alter the functional properties ofthe nucleic acid or the encoded polypeptide. Due to the degeneracy ofthe genetic code, a base pair change can result in no change in theencoded amino acid sequence. For example, the codons ACT, ACC, ACA andACG all encode a threonine amino acid. Alternatively one or more basepair changes may alter the encoded amino acid however if the substitutedamino acid has similar chemical properties functionality of the encodedprotein is likely to be unaffected. For example, threonine codons ACTand ACC when changed to AGT or AGC respectively encode for serine, achemically and biologically similar amino acid. Additionally, certainamino acids within a polypeptide are non essential and alterations maybe made in these locations without an effect on the functionality of thepolypeptide. Substantially similar also refers to sequences havingchanges at one or more nucleotide bases wherein the changes do notaffect the ability of the sequence to alter gene expression by variousgene silencing methodologies such as antisense, RNAi or co-suppression.The term “substantially similar” refers to polypeptides wherein a changein one or more amino acids does not alter the functional properties ofthe polypeptide as discussed above.

The term “yield” refers to seed number, seed weight, seed size, totalplant biomass, increased biomass of a plant organ, such as stems orleaves or roots, fruit production, and flower production,

The term “yield protection” is defined as the positive difference,expressed as a % value, between the yield of the transgenic or mutantand the control, where the yield is expressed as a % of optimal,following an imposed stress. The calculation is done by comparing theoptimal yield with that after the stress treatment (stress yield/optimalyield×100).

The MYB gene family is classified based on sequence homology and thepresence of defined domains and motifs such as an R2R3 domain. Theclassification in all cases is not absolute and varies depending on thecriteria selected for the analysis (Krantz et al 1998, Stracke et al2001).

Herein we define the MYB-subgroup14 to include at least the followingmembers, MYB68, MYB36, MYB84, MYB37, MYB38 and MYB87. The ArabidopsisMYB68, MYB36, MYB84, MYB37, MYB38 and MYB87 sequences are used toidentify homologues from other species according to the methods herein,examples of which are included in Table 1

The term “MYB sequence” refers to a polynucleotide sequence or apolypeptide sequence as contextually appropriate.

Sequences

The following sequences from the MYB-subgroup14 family, andcorresponding sequence identifiers, are employed throughout thespecification, examples and appended claims:

TABLE 1 SEQ ID Accession Number MYB NO: SPECIES Reference Identification1 ARABIDOPSIS THALIANA NM_125976.2 MYB68 NT 2 ARABIDOPSIS THALIANANP_201380.1 MYB68 AA 3 ARABIDOPSIS THALIANA NM_114829.3 MYB84 NT 4ARABIDOPSIS THALIANA NP_190538.1 MYB84 AA 5 ARABIDOPSIS THALIANANM_125143.3 MYB36 NT 6 ARABIDOPSIS THALIANA NP_200570.1 MYB36 AA 7BRASSICA RAPA MYB68 NT 8 BRASSICA RAPA MYB68 AA 9 ORYZA SATIVANM_001057941.1 MYB36 NT 10 ORYZA SATIVA AAT85046.1 MYB36 AA 11 GOSSYPIUMTC34239 MYB68 NT 12 GOSSYPIUM TC34239_ORF MYB68 AA 13 GLYCINE MAXDQ822965.1 MYB84 NT 14 GLYCINE MAX ABH02906.1 MYB84 AA 15 GLYCINE MAXMYB84 NT 16 GLYCINE MAX MYB84 AA 17 ZEA MAYS TC370133 MYB84 NT 18 ZEAMAYS TC370133_ORF MYB84 AA 19 SORGHUM BICOLOR AF474127 MYB36 NT 20SORGHUM BICOLOR AAL84760.1 MYB36 AA 21 TRITICUM AESTIVUM BQ483726 MYB84NT 22 TRITICUM AESTIVUM BQ483726_ORF MYB84 AA 23 POPULUS TC54478 MYB84NT 24 POPULUS TC54478_ORF MYB84 AA 25 MEDICAGO TC97441 MYB68 NTTRUNCATULA 26 MEDICAGO TC97441_ORF MYB68 AA TRUNCATULA 27 SOLANUMAF426174.1 MYB36 NT LYCOPERSICUM 28 SOLANUM AAL69334.1 MYB36 AALYCOPERSICUM 29 SOLANUM BG134669 MYB36 NT LYCOPERSICUM 30 SOLANUMBG134669_ORF MYB36 AA LYCOPERSICUM 31 ARABIDOPSIS THALIANA NM_119940.3MYB87 NT 32 ARABIDOPSIS THALIANA NP_195492.2 MYB87 AA 33 ARABIDOPSISTHALIANA NM_122206.3 MYB37 NT 34 ARABIDOPSIS THALIANA NP_197691.1 MYB37AA 35 ARABIDOPSIS THALIANA NM_129245.2 MYB38 NT 36 ARABIDOPSIS THALIANANP_181226.1 MYB38 AA 37 AEGILOPS SPELTOIDES BQ841600.1 MYB36 NT 38ANTIRRHINUM MAJUS AJ794728.1 MYB68 NT 39 ANTIRRHINUM MAJUSAJ794728.1_ORF MYB68 AA 40 AQUILEGIA TC13008 MYB84 NT 41 AQUILEGIATC13008_ORF MYB84 AA 42 AQUILEGIA TC11167 MYB36 NT 43 AQUILEGIATC11167_ORF MYB36 AA 44 ARACHIS HYPOGAEA CD038321.1 MYB68 NT 45 ARACHISHYPOGAEA CD038321.1_ORF MYB68 AA 46 ARACHIS HYPOGAEA ES761155.1 MYB68 NT47 ARACHIS HYPOGAEA ES761155.1_ORF MYB68 AA 48 ARACHIS STENOSPERMAEH046152.1 MYB36 NT 49 ARACHIS STENOSPERMA EH046152.1_ORF MYB36 AA 50BRACHYPODIUM DV486330.1 MYB38 NT DISTACHYON 51 BRACHYPODIUMDV486330.1_ORF MYB38 AA DISTACHYON 52 BRACHYPODIUM DV488965.1 MYB38 NTDISTACHYON 53 BRACHYPODIUM DV488965.1_ORF MYB38 AA DISTACHYON 54BRASSICA NAPUS (bud) MYB68 NT 55 BRASSICA NAPUS (bud) MYB68 AA 56BRASSICA NAPUS (root) MYB68 NT 57 BRASSICA NAPUS (root) MYB68 AA 58BRASSICA NAPUS TC40384 MYB68 NT 59 BRASSICA NAPUS ES900275.1 MYB68 NT 60BRASSICA NAPUS ES900275.1_ORF MYB68 AA 61 BRASSICA NAPUS TC55899 MYB38NT 62 BRASSICA NAPUS TC55899_ORF MYB38 AA 63 BRASSICA RAPA EX134980.1MYB68 NT 64 BRASSICA RAPA EX134980.1_ORF MYB68 AA 65 BRASSICA RAPAEX137439.1 MYB68 NT 66 BRASSICA RAPA EX137439.1_ORF MYB68 AA 67CARTHAMUS EL384492.1 MYB36 NT TINCTORIUS 68 CARTHAMUS EL384492.1_ORFMYB36 AA TINCTORIUS 69 CARTHAMUS EL392277.1 MYB36 NT TINCTORIUS 70CARTHAMUS EL392277.1_ORF MYB36 AA TINCTORIUS 71 CENTAUREA MACULOSAEH724496.1 MYB36 NT 72 CENTAUREA MACULOSA EH724496.1_ORF MYB36 AA 73CENTAUREA MACULOSA EH719165.1 MYB36 NT 74 CENTAUREA MACULOSAEH719165.1_ORF MYB36 AA 75 CENTAUREA MACULOSA EH724438.1 MYB68 NT 76CENTAUREA MACULOSA EH724438.1_ORF MYB68 AA 77 CENTAUREA EH774519.1 MYB68NT SOLSTITIALIS 78 CENTAUREA EH774519.1_ORF MYB68 AA SOLSTITIALIS 79CENTAUREA EH771972.1 MYB68 NT SOLSTITIALIS 80 CENTAUREA EH771972.1_ORFMYB68 AA SOLSTITIALIS 81 CENTAUREA EH768792.1 MYB84 NT SOLSTITIALIS 82CENTAUREA EH768792.1_ORF MYB84 AA SOLSTITIALIS 83 CICHORIUM ENDIVIAEL361859.1 MYB84 NT 84 CICHORIUM ENDIVIA EL361859.1_ORF MYB84 AA 85CICHORIUM INTYBUS EH681135.1 MYB38 NT 86 CICHORIUM INTYBUSEH681135.1_ORF MYB38 AA 87 CICHORIUM INTYBUS EH694860.1 MYB68 NT 88CITRUS SINENSIS CK936024.1 MYB68 NT 89 CITRUS SINENSIS CK936024.1_ORFMYB68 AA 90 COFFEA CANEPHORA DV692261.1 MYB37 NT 91 COFFEA CANEPHORADV691112.1 MYB37 NT 92 CUCUMIS MELO AM727197.2 MYB36 NT 93 CUCUMIS MELOAM727197.2_ORF MYB36 AA 94 CUCUMIS MELO AM716075.2 MYB36 NT 95 CUCUMISMELO AM716075.2_ORF MYB36 AA 96 DAUCUS CAROTA AB298508.1 MYB68 NT 97DAUCUS CAROTA BAF49444.1 MYB68 AA 98 ELAEIS GUINEENSIS EL690464.1 MYB84NT 99 ELAEIS GUINEENSIS EL690464.1_ORF MYB84 AA 100 ELAEIS OLEIFERAES370938.1 MYB84 NT 101 ELAEIS OLEIFERA ES370938.1_ORF MYB84 AA 102ESCHSCHOLZIA CD480801.1 MYB68 NT CALIFORNICA 103 ESCHSCHOLZIACD480801.1_ORF MYB68 AA CALIFORNICA 104 EUPHORBIA ESULA DV138530.1 MYB84NT 105 EUPHORBIA ESULA DV138530.1_ORF MYB84 AA 106 EUPHORBIA ESULADV126436.1 MYB36 NT 107 EUPHORBIA ESULA DV126436.1_ORF MYB36 AA 108EUPHORBIA TIRUCALLI BP958179.1 MYB84 NT 109 GINKGO BILOBA EX940876.1MYB68 NT 110 GLYCINE MAX MYB84 NT 111 GLYCINE MAX MYB84 AA 112 GLYCINEMAX TC213651 MYB84 NT 113 GLYCINE MAX TC213651_ORF MYB84 AA 114 GLYCINEMAX DQ822971.1 MYB36 NT 115 GLYCINE MAX ABH02912.1 MYB36 AA 116 GLYCINEMAX TC211227 MYB36 NT 117 GLYCINE MAX TC211227_ORF MYB36 AA 118GOSSYPIUM TC62721 MYB68 NT 119 GOSSYPIUM TC62721_ORF MYB68 AA 120GOSSYPIUM DW491290.1 MYB36 NT 121 GOSSYPIUM DW491290.1_ORF MYB36 AA 122HEDYOTIS TERMINALIS CB077617.1 MYB84 NT 123 HEDYOTIS TERMINALISCB077617.1_ORF MYB84 AA 124 HELIANTHUS ANNUUS BQ967558 MYB36 NT 125HELIANTHUS EE621630.1 MYB36 NT ARGOPHYLLUS 126 HELIANTHUS EE621630.1_ORFMYB36 AA ARGOPHYLLUS 127 HELIANTHUS EE619500.1 MYB36 NT ARGOPHYLLUS 128HELIANTHUS EE619500.1_ORF MYB36 AA ARGOPHYLLUS 129 HELIANTHUS CILIARISEL422629.1 MYB68 NT 130 HELIANTHUS EXILIS EE645503.1 MYB68 NT 131HELIANTHUS EXILIS EE645503.1_ORF MYB68 AA 132 HELIANTHUS EXILISEE646813.1 MYB36 NT 133 HELIANTHUS EXILIS EE646813.1_ORF MYB36 AA 134HELIANTHUS EL474327.1 MYB84 NT PARADOXUS 135 HELIANTHUS PETIOLARISDY942970.1 MYB84 NT 136 HELIANTHUS PETIOLARIS DY942970.1_ORF MYB84 AA137 HELIANTHUS PETIOLARIS DY953493.1 MYB68 NT 138 HELIANTHUS PETIOLARISDY953493.1_ORF MYB68 AA 139 HELIANTHUS EL445341.1 MYB36 NT TUBEROSUS 140HELIANTHUS EL445341.1_ORF MYB36 AA TUBEROSUS 141 HORDEUM VULGAREBY845215.1 MYB38 NT 142 HORDEUM VULGARE BY845215.1_ORF MYB38 AA 143HUMULUS LUPULUS AJ876882.1 MYB36 NT 144 HUMULUS LUPULUS CAI46244.1 MYB36AA 145 LACTUCA PERENNIS DW092247.1 MYB84 NT 146 LACTUCA PERENNISDW092247.1_ORF MYB84 AA 147 LACTUCA SALIGNA DW065247.1 MYB68 NT 148LACTUCA SALIGNA DW065247.1_ORF MYB68 AA 149 LACTUCA SATIVA DY960463.1MYB38 NT 150 LACTUCA SATIVA DY960463.1_ORF MYB38 AA 151 LACTUCA SATIVADY969483.1 MYB38 NT 152 LACTUCA SATIVA DY969483.1_ORF MYB38 AA 153LACTUCA SATIVA DY980672.1 MYB38 NT 154 LACTUCA SATIVA DY980672.1_ORFMYB38 AA 155 LACTUCA SERRIOLA DW108054.1 MYB38 NT 156 LACTUCA VIROSADW160139.1 MYB84 NT 157 LACTUCA VIROSA DW160139.1_ORF MYB84 AA 158LACTUCA VIROSA DW160891.1 MYB38 NT 159 LACTUCA VIROSA DW160891.1_ORFMYB38 AA 160 LIRIODENDRON CO998829.1 MYB38 NT TULIPIFERA 161LIRIODENDRON CO998829.1_ORF MYB38 AA TULIPIFERA 162 MALUS DOMESTICADT002401.1 MYB36 NT 163 MALUS DOMESTICA DT002401.1_ORF MYB36 AA 164MALUS DOMESTICA DQ074472.1 MYB38 NT 165 MALUS DOMESTICA AAZ20440.1 MYB38AA 166 MANIHOT ESCULENTA DB936694.1 MYB68 NT 167 MANIHOT ESCULENTADB936694.1_ORF MYB68 AA 168 MARCHANTIA BJ846153.1 MYB84 NT POLYMORPHA169 MARCHANTIA BJ846153.1_ORF MYB84 AA POLYMORPHA 170 MEDICAGO TC110497MYB36 NT TRUNCATULA 171 MEDICAGO TC110497_ORF MYB36 AA TRUNCATULA 172MEDICAGO BF634640 MYB84 NT TRUNCATULA 173 MEDICAGO BF634640_ORF MYB84 AATRUNCATULA 174 NUPHAR ADVENA CD472544.1 MYB36 NT 175 NUPHAR ADVENACD472544.1_ORF MYB36 AA 176 ORYZA SATIVA LOC_Os01g09590.1_cds MYB38 NT177 ORYZA SATIVA LOC_Os01g09590.1 MYB38 AA 178 ORYZA SATIVALOC_Os01g49160.1_cds MYB36 NT 179 ORYZA SATIVA LOC_Os01g49160.1 MYB36 AA180 ORYZA SATIVA LOC_Os01g52410.1_cds MYB38 NT 181 ORYZA SATIVALOC_Os01g52410.1 MYB38 AA 182 ORYZA SATIVA LOC_Os02g54520.1_cds MYB36 NT183 ORYZA SATIVA LOC_Os02g54520.1 MYB36 AA 184 ORYZA SATIVALOC_Os05g48010.1_cds MYB36 NT 185 ORYZA SATIVA LOC_Os05g48010.1 MYB36 AA186 ORYZA SATIVA LOC_Os08g15020.1_cds MYB36 NT 187 ORYZA SATIVALOC_Os08g15020.1 MYB36 AA 188 ORYZA SATIVA LOC_Os09g26170.1_cds MYB36 NT189 ORYZA SATIVA LOC_Os09g26170.1 MYB36 AA 190 ORYZA SATIVALOC_Os10g35660.1_cds MYB36 NT 191 ORYZA SATIVA LOC_Os10g35660.1 MYB36 AA192 PICEA EX361512.1 MYB68 NT 193 PICEA EX361512.1_ORF MYB68 AA 194PICEA TC20498 MYB68 NT 195 PICEA TC20498_ORF MYB68 AA 196 PINUS DR015810MYB84 NT 197 PINUS DR015810_ORF MYB84 AA 198 PINUS TC66643 MYB68 NT 199PINUS TC66643_ORF MYB68 AA 200 PONCIRUS TRIFOLIATA CD575120.1 MYB38 NT201 PONCIRUS TRIFOLIATA CD575120.1_ORF MYB38 AA 202 POPULUS Gw1.II.96.1MYB68 NT 203 POPULUS Gw1.II.96.1_ORF MYB68 AA 204 POPULUS DB879439.1MYB84 NT 205 POPULUS DB879439.1_ORF MYB84 AA 206 POPULUS TC74579 MYB36NT 207 POPULUS TC74579_ORF MYB36 AA 208 QUERCUS PETRAEA CU639795.1 MYB36NT 209 QUERCUS PETRAEA CU639795.1_ORF MYB36 AA 210 QUERCUS SUBEREE743680.1 MYB84 NT 211 RAPHANUS FD544184.1 MYB68 NT RAPHANISTRUM 212RAPHANUS FD544184.1_ORF MYB68 AA RAPHANISTRUM 213 RAPHANUS EY915531.1MYB68 NT RAPHANISTRUM 214 RAPHANUS FD540311.1 MYB68 NT RAPHANISTRUM 215RAPHANUS FD540311.1_ORF MYB68 AA RAPHANISTRUM 216 RAPHANUS EV548164.1MYB38 NT RAPHANISTRUM 217 RAPHANUS SATIVUS FD580369.1 MYB68 NT 218 ROSAHYBRID EC587279.1 MYB68 NT 219 ROSA HYBRID EC587279.1_ORF MYB68 AA 220SACCHARUM CA150911 MYB36 NT OFFICINARUM 221 SACCHARUM CA150911_ORF MYB36AA OFFICINARUM 222 SACCHARUM CA258665 MYB84 NT OFFICINARUM 223 SACCHARUMCA258665_ORF MYB84 AA OFFICINARUM 224 SACCHARUM TC44677 MYB36 NTOFFICINARUM 225 SACCHARUM TC44677_ORF MYB36 AA OFFICINARUM 226 SECALECEREALE BE495537 MYB38 NT 227 SECALE CEREALE BE495537_ORF MYB38 AA 228SOLANUM TC182203 MYB36 NT LYCOPERSICUM 229 SOLANUM TC182203_ORF MYB36 AALYCOPERSICUM 230 SOLANUM TUBEROSUM AM907873.1 MYB36 NT 231 SOLANUMTUBEROSUM AM907873.1_ORF MYB36 AA 232 SORGHUM BICOLOR TC98185 MYB36 NT233 SORGHUM BICOLOR TC98185_ORF MYB36 AA 234 SORGHUM BICOLOR TC101637MYB36 NT 235 SORGHUM BICOLOR TC101637_ORF MYB36 AA 236 SORGHUMPROPINQUUM BG560270.1 MYB36 NT 237 SORGHUM PROPINQUUM BG560270.1_ORFMYB36 AA 238 TARAXACUM DY830100.1 MYB68 NT OFFICINALE 239 TARAXACUMDY830100.1_ORF MYB68 AA OFFICINALE 240 TRIPHYSARIA PUSILLA EY172046.1MYB68 NT 241 TRIPHYSARIA PUSILLA EY172046.1_ORF MYB68 AA 242 TRIPHYSARIAPUSILLA EY179359.1 MYB36 NT 243 TRIPHYSARIA PUSILLA EY179359.1_ORF MYB36AA 244 TRIPHYSARIA PUSILLA EY174724.1 MYB38 NT 245 TRIPHYSARIA PUSILLAEY174724.1_ORF MYB38 AA 246 TRIPHYSARIA EX989121.1 MYB38 NT VERSICOLOR247 TRIPHYSARIA EY018825.1 MYB38 NT VERSICOLOR 248 TRIPHYSARIAEY018825.1_ORF MYB38 AA VERSICOLOR 249 TRITICUM AESTIVUM MYB84 NT 250TRITICUM AESTIVUM MYB84 AA 251 VACCINIUM CV090776.1 MYB36 NT CORYMBOSUM252 VACCINIUM CV090776.1_ORF MYB36 AA CORYMBOSUM 253 VITIS VINIFERACAO70108.1_cds MYB84 NT 254 VITIS VINIFERA CAO70108.1 MYB84 AA 255 VITISVINIFERA CAO43296.1_cds MYB36 NT 256 VITIS VINIFERA CAO43296.1 MYB36 AA257 VITIS VINIFERA CAO61524.1_cds MYB84 NT 258 VITIS VINIFERA CAO61524.1MYB84 AA 259 VITIS VINIFERA DT006424 MYB36 NT 260 VITIS VINIFERADT006424_ORF MYB36 AA 261 ZEA MAYS MYB36 NT 262 ZEA MAYS MYB36 AA 263ZEA MAYS TC320820 MYB36 NT 264 ZEA MAYS TC320820_ORF MYB36 AA 265 ORYZASATIVA MYB36 NT

Determining Homology Between Two or More Sequences

To determine the percent homology of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in either of the sequences being comparedfor optimal alignment between the sequences). The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules arehomologous at that position (i.e., as used herein amino acid or nucleicacid “homology” is equivalent to amino acid or nucleic acid “identity”).

The nucleic acid sequence homology may be determined as the degree ofidentity between two sequences. The homology may be determined usingcomputer programs known in the art, such as GAP software provided in theGCG program package. See, Needleman and Wunsch 1970 J Mol Biol 48:443-453. Using GCG GAP software with the following settings for nucleicacid sequence comparison: GAP creation penalty of 5.0 and GAP extensionpenalty of 0.3, the coding region of the analogous nucleic acidsequences referred to above exhibits a degree of identity preferably ofat least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, with the codingsequence (encoding) part of the DNA sequence shown in Table 1.

The term “sequence identity” refers to the degree to which twopolynucleotide or polypeptide sequences are identical on aresidue-by-residue basis over a particular region of comparison. Theterm “percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over that region of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U, or I, in the case of nucleic acids) occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the region ofcomparison (i.e., the window size), and multiplying the result by 100 toyield the percentage of sequence identity. The term “substantialidentity” as used herein denotes a characteristic of a polynucleotidesequence, wherein the polynucleotide comprises a sequence that has atleast 80 percent sequence identity, preferably at least 85 percentidentity and often 90 to 95 percent sequence identity, more usually atleast 99 percent sequence identity as compared to a reference sequenceover a comparison region. The term “percentage of positive residues” iscalculated by comparing two optimally aligned sequences over that regionof comparison, determining the number of positions at which theidentical and conservative amino acid substitutions, as defined above,occur in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the region of comparison (i.e., the window size), andmultiplying the result by 100 to yield the percentage of positiveresidues.

Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding a MYB-subgroup14protein, gene, analogs or homologs thereof. The sequence encoding aMYB-subgroup14 polypeptide may be a genomic sequence or a cDNA sequence.As used herein the term expression vector includes vectors which aredesigned to provide transcription of the nucleic acid sequence. Thetranscribed nucleic acid may be translated into a polypeptide or proteinproduct. As used herein, the term “vector” refers to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments canbe ligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication). Other vectors are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively-linked.Such vectors are referred to herein as “expression vectors”. In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” can be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors or planttransformation vectors, binary or otherwise, which serve equivalentfunctions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, that is operatively-linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably-linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerthat allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell).

The term “regulatory sequence” is intended to include promoters,enhancers and other expression control elements (e.g., polyadenylationsignals). Such regulatory sequences are described, for example, inGoeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, AcademicPress, San Diego, Calif. (1990). Regulatory sequences include those thatdirect constitutive expression of a nucleotide sequence in many types ofhost cell and those that direct expression of the nucleotide sequenceonly in certain host cells (e.g., tissue-specific regulatory sequences)or inducible promoters (e.g., induced in response to abiotic factorssuch as environmental conditions, heat, drought, nutrient status orphysiological status of the cell or biotic such as pathogen responsive).Examples of suitable promoters include for example constitutivepromoters, ABA inducible promoters, tissue specific promoters andabiotic or biotic inducible promoters. It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired as well as timing and location ofexpression, etc. The expression vectors of the invention can beintroduced into host cells to thereby produce proteins or peptides,including fusion proteins or peptides, encoded by nucleic acids asdescribed herein (e.g., MYB-subgroup14 proteins such as MYB68 proteins,mutant forms of MYB68 proteins, fusion proteins, etc.).

The recombinant expression vectors of the invention can be designed forexpression of a MYB-subgroup14 gene or a MYB-subgroup14 protein inprokaryotic or eukaryotic cells. For example, a MYB-subgroup14 gene or aMYB-subgroup14 protein can be expressed in bacterial cells such asEscherichia coli, insect cells (using baculovirus expression vectors)yeast cells, plant cells or mammalian cells. Suitable host cells arediscussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS INENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively,the recombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

In one embodiment, a nucleic acid of the invention is expressed inplants cells using a plant expression vector. Examples of plantexpression vectors systems include tumor inducing (Ti) plasmid orportion thereof found in Agrobacterium, cauliflower mosaic virus (CAMV)DNA and vectors such as pBI121, a pCAMBA series vector or one ofpreferred choice to a person skilled in the art.

For expression in plants, the recombinant expression cassette willcontain in addition to a MYB-subgroup14 polynucleotide, a promoterregion functional in a plant cell, a transcription initiation site (ifthe coding sequence to transcribed lacks one), and a transcriptiontermination/polyadenylation sequence. The termination/polyadenylationregion may be obtained from the same gene as the promoter sequence ormay be obtained from different genes. Unique restriction enzyme sites atthe 5′ and 3′ ends of the cassette are typically included to allow foreasy insertion into a pre-existing vector.

Examples of suitable plant expressible promoters include promoters fromplant viruses such as the 35S promoter from cauliflower mosaic virus(CaMV) (Odell, et al., Nature, 313: 810-812 (1985)), promoters fromgenes such as rice actin (McElroy, et al., Plant Cell, 163-171 (1990)),ubiquitin (Christensen, et al., Plant Mol. Biol., 12: 619-632 (1992);and Christensen, et al., Plant Mol. Biol., 18: 675-689 (1992)), pEMU(Last, et al., Theor. Appl. Genet., 81: 581-588 (1991)), MAS (Velten, etal., EMBO J., 3: 2723-2730 (1984)), maize H3 histone (Lepetit, et al.,Mol. Gen. Genet., 231: 276-285 (1992); and Atanassvoa, et al., PlantJournal, 2(3): 291-300 (1992)), the 5′- or 3′-promoter derived fromT-DNA of Agrobacterium tumefaciens, the Smas promoter, the cinnamylalcohol dehydrogenase promoter (U.S. Pat. No. 5,683,439), the Nospromoter, the rubisco promoter, the GRP1-8 promoter, ALS promoter, (WO96/30530), a synthetic promoter, such as Rsyn7, SCP and UCP promoters,ribulose-1,3-diphosphate carboxylase, fruit-specific promoters, heatshock promoters, seed-specific promoters and other transcriptioninitiation regions from various plant genes, for example, including thevarious opine initiation regions, such as for example, octopine,mannopine, and nopaline. Useful promoters also include heat induciblepromoters such as the HSP18.2 or HSP81.1 promoters (Takahashi et al.1992, Plant J. 2, 751-761; Yoshida et al., 1995, Appl. Microbiol.Biotechnol. 44, 466-472; Ueda et al., 1996, Mol Gen Genet. 250,533-539). Cryptic promoters are also useful for chimeric constructsuseful in the invention. Cryptic gene regulatory elements are inactiveat their native locations in the genome but are fully functional whenpositioned adjacent to genes in transgenic plants.

In addition to chimeric promoter-gene constructs the use of a nativeMYB-subgroup14 promoter is contemplated. Expression characteristics of anative promoter may be modified by inclusion of regulatory elements suchthat expression levels are elevated and or expressed ectopically and orconstitutively. For example, a 4×35S enhancer sequence (Wiegel et al.,2000) may be included in a construct to enhance expression.Alternatively a population of plants may be produced by transformationwith a construct having a 4×35S enhancer sequence, such as, a pSKI15vector as per Wiegel et al., 2000. The transformed population can bescreened for plants having increased expression of a MYB-subgroup14sequence, or screened for plants having increased heat tolerance andreduced flower abortion, or a combination of such screens to identify aplant of interest.

Additional regulatory elements that may be connected to a MYB-subgroup14encoding nucleic acid sequence for expression in plant cells includeterminators, polyadenylation sequences, and nucleic acid sequencesencoding signal peptides that permit localization within a plant cell orsecretion of the protein from the cell. Such regulatory elements andmethods for adding or exchanging these elements with the regulatoryelements of a MYB-subgroup14 gene are known, and include, but are notlimited to, 3′ termination and/or polyadenylation regions such as thoseof the Agrobacterium tumefaciens nopaline synthase (nos) gene (Bevan, etal., Nucl. Acids Res., 12: 369-385 (1983)); the potato proteinaseinhibitor II (PINII) gene (Keil, et al., Nucl. Acids Res., 14: 5641-5650(1986) and hereby incorporated by reference); and An, et al., PlantCell, 1: 115-122 (1989)); and the CaMV 19S gene (Mogen, et al., PlantCell, 2: 1261-1272 (1990)).

Plant signal sequences, including, but not limited to, signal-peptideencoding DNA/RNA sequences which target proteins to the extracellularmatrix of the plant cell (Dratewka-Kos, et al., J. Biol. Chem., 264:4896-4900 (1989)) and the Nicotiana plumbaginifolia extension gene(DeLoose, et al., Gene, 99: 95-100 (1991)), or signal peptides whichtarget proteins to the vacuole like the sweet potato sporamin gene(Matsuka, et al., Proc. Nat'l Acad. Sci. (USA), 88: 834 (1991)) and thebarley lectin gene (Wilkins, et al., Plant Cell, 2: 301-313 (1990)), orsignals which cause proteins to be secreted such as that of PRIb (Lind,et al., Plant Mol. Biol., 18: 47-53 (1992)), or those which targetproteins to the plastids such as that of rapeseed enoyl-ACP reductase(Verwaert, et al., Plant Mol. Biol., 26: 189-202 (1994)) are useful inthe invention.

In another embodiment, the recombinant expression vector is capable ofdirecting expression of the nucleic acid preferentially in a particularcell type (e.g., tissue-specific regulatory elements are used to expressthe nucleic acid). Tissue-specific regulatory elements are known in theart. Especially useful in connection with the nucleic acids of thepresent invention are expression systems which are operable in plants.These include systems which are under control of a tissue-specificpromoter, as well as those which involve promoters that are operable inall plant tissues.

Organ-specific promoters are also well known. For example, the chalconesynthase-A gene (van der Meer et al., 1990, Plant Molecular Biology15(1):95-109) or the dihydroflavonol-4-reductase (dfr) promoter (Elomaaet al., The Plant Journal, 16(1) 93-99) direct expression in specificfloral tissues. Also available are the patatin class I promoter istranscriptionally activated only in the potato tuber and can be used totarget gene expression in the tuber (Bevan, M., 1986, Nucleic AcidsResearch 14:4625-4636). Another potato-specific promoter is thegranule-bound starch synthase (GBSS) promoter (Visser, R. G. R, et al.,1991, Plant Molecular Biology 17:691-699).

Other organ-specific promoters appropriate for a desired target organcan be isolated using known procedures. These control sequences aregenerally associated with genes uniquely expressed in the desired organ.In a typical higher plant, each organ has thousands of mRNAs that areabsent from other organ systems (reviewed in Goldberg, P., 1986, Trans.R. Soc. London B314:343).

The resulting expression system or cassette is ligated into or otherwiseconstructed to be included in a recombinant vector which is appropriatefor plant transformation. The vector may also contain a selectablemarker gene by which transformed plant cells can be identified inculture. The marker gene may encode antibiotic resistance. These markersinclude resistance to G418, hygromycin, bleomycin, kanamycin, andgentamicin. Alternatively the marker gene may encode a herbicidetolerance gene that provides tolerance to glufosinate or glyphosate typeherbicides. After transforming the plant cells, those cells having thevector will be identified by their ability to grow on a mediumcontaining the particular antibiotic or herbicide. Replicationsequences, of bacterial or viral origin, are generally also included toallow the vector to be cloned in a bacterial or phage host, preferably abroad host range prokaryotic origin of replication is included. Aselectable marker for bacteria should also be included to allowselection of bacterial cells bearing the desired construct. Suitableprokaryotic selectable markers also include resistance to antibioticssuch as kanamycin or tetracycline.

Other DNA sequences encoding additional functions may also be present inthe vector, as is known in the art. For instance, in the case ofAgrobacterium transformations, T-DNA sequences will also be included forsubsequent transfer to plant chromosomes.

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but also to the progeny or potential progeny ofsuch a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein. Vector DNA can beintroduced into prokaryotic or eukaryotic cells via conventionaltransformation or transfection techniques. As used herein, the terms“transformation” and “transfection” are intended to refer to a varietyof art-recognized techniques for introducing foreign nucleic acid (e.g.,DNA) into a host cell.

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) a polypeptide ofthe invention encoded in an open reading frame of a polynucleotide ofthe invention. Accordingly, the invention further provides methods forproducing a polypeptide using the host cells of the invention. In oneembodiment, the method comprises culturing the host cell of invention(into which a recombinant expression vector encoding a polypeptide ofthe invention has been introduced) in a suitable medium such that thepolypeptide is produced. In another embodiment, the method furthercomprises isolating the polypeptide from the medium or the host cell.

A number of cell types may act as suitable host cell for expression of apolypeptide encoded by an open reading frame in a polynucleotide of theinvention. Plant host cells include, for example, plant cells that couldfunction as suitable hosts for the expression of a polynucleotide of theinvention include epidermal cells, mesophyll and other ground tissues,and vascular tissues in leaves, stems, floral organs, and roots from avariety of plant species, for example Arabidopsis, Brassica, Oryza, Zea,Sorghum, Gossypium, Triticum, Glycine, Pisum, Phaseolus, Lycopersicon,Trifolium, Cannabis, Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus,Medicago, Onobrychis, Trigonella, Vigna, Citrus, Linum, Geranium,Manihot, Daucus, Raphanus, Sinapis, Atropa, Capsicum, Datura,Hyoscyamus, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Ciahorium,Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis,Nemesis, Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio,Salpiglossis, Cucumis, Browaalia, Lolium, Avena, Hordeum, Secale, Picea,Caco, and Populus.

Conservative Mutations

In addition to naturally-occurring allelic variants of a MYB-subgroup14or a MYB68 sequence that may exist in the population, the skilledartisan will further appreciate that changes can be introduced bymutation into the nucleotide sequence of SEQ ID NO:1, 3, 5, 7, 9, 11,13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 38, 40, 42, 44, 46,48, 50, 52, 54, 56, 58, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81,83, 85, 87, 88, 90, 91, 92, 94, 96, 98, 100, 102, 104, 106, 108, 109,110, 112, 114, 116, 118, 120, 122, 124, 125, 127, 129, 130, 132, 134,135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 156, 158, 160,162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188,190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 211, 213, 214,216, 217, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240,242, 244, 246, 247, 249, 251, 253, 255, 257, 259, 261, 263 and 265thereby leading to changes in the amino acid sequence of the encodedMYB-subgroup14 or a MYB68 protein, without altering the functionalability of the MYB-subgroup14 or a MYB68 protein. For example,nucleotide substitutions leading to amino acid substitutions at“non-essential” amino acid residues can be made in the sequence of SEQID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,36, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 60, 62, 64, 66, 68, 70, 72,74, 76, 78, 80, 82, 84, 86, 89, 93, 95, 97, 99, 101, 103, 105, 107, 111,113, 115, 117, 119, 121, 123, 126, 128, 131, 133, 136, 138, 140, 142,144, 146, 148, 150, 152, 154, 157, 159, 161, 163, 165, 167, 169, 171,173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199,201, 203, 205, 207, 209, 212, 215, 219, 221, 223, 225, 227, 229, 231,233, 235, 237, 239, 241, 243, 245, 248, 250, 252, 254, 256, 258, 260,262 and 264. A “non-essential” amino acid residue is a residue that canbe altered from the wild-type sequence of a MYB-subgroup14 or a MYB68without altering the biological activity, whereas an “essential” aminoacid residue is required for biological activity. For example, aminoacid residues that are conserved among MYB-subgroup14 or MYB68 proteinsof the present invention are predicted to be poor candidates foralteration. Alignments and identification of conserved regions aredescribed herein and provide further guidance as to identification ofessential amino acids and conserved amino acids.

Another aspect of the invention pertains to nucleic acid moleculesencoding a MYB-subgroup14 or MYB68 protein that contain changes in aminoacid residues that are not essential for activity. Such MYB-subgroup14or MYB68 proteins differ in amino acid sequence from SEQ ID NO: 2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 39, 41, 43,45, 47, 49, 51, 53, 55, 57, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80,82, 84, 86, 89, 93, 95, 97, 99, 101, 103, 105, 107, 111, 113, 115, 117,119, 121, 123, 126, 128, 131, 133, 136, 138, 140, 142, 144, 146, 148,150, 152, 154, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177,179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205,207, 209, 212, 215, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237,239, 241, 243, 245, 248, 250, 252, 254, 256, 258, 260, 262 and 264 yetretain biological activity. In one embodiment, the isolated nucleic acidmolecule comprises a nucleotide sequence encoding a protein, wherein theprotein comprises an amino acid sequence at least about 75% homologousto the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 39, 41, 43, 45, 47, 49, 51, 53, 55,57, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 89, 93, 95,97, 99, 101, 103, 105, 107, 111, 113, 115, 117, 119, 121, 123, 126, 128,131, 133, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 157, 159,161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187,189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 212, 215, 219,221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 248,250, 252, 254, 256, 258, 260, 262 and 264. Preferably, the proteinencoded by the nucleic acid is at least about 80% homologous to SEQ IDNO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 60, 62, 64, 66, 68, 70, 72, 74,76, 78, 80, 82, 84, 86, 89, 93, 95, 97, 99, 101, 103, 105, 107, 111,113, 115, 117, 119, 121, 123, 126, 128, 131, 133, 136, 138, 140, 142,144, 146, 148, 150, 152, 154, 157, 159, 161, 163, 165, 167, 169, 171,173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199,201, 203, 205, 207, 209, 212, 215, 219, 221, 223, 225, 227, 229, 231,233, 235, 237, 239, 241, 243, 245, 248, 250, 252, 254, 256, 258, 260,262 and 264 more preferably at least about 90%, 95%, 98%, and mostpreferably at least about 99% homologous to SEQ ID NO: 2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 39, 41, 43, 45, 47,49, 51, 53, 55, 57, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84,86, 89, 93, 95, 97, 99, 101, 103, 105, 107, 111, 113, 115, 117, 119,121, 123, 126, 128, 131, 133, 136, 138, 140, 142, 144, 146, 148, 150,152, 154, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179,181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207,209, 212, 215, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239,241, 243, 245, 248, 250, 252, 254, 256, 258, 260, 262 and 264.

An isolated nucleic acid molecule encoding a MYB-subgroup14 or a MYB68protein homologous to the protein of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 39, 41, 43, 45, 47, 49, 51,53, 55, 57, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 89,93, 95, 97, 99, 101, 103, 105, 107, 111, 113, 115, 117, 119, 121, 123,126, 128, 131, 133, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154,157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183,185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 212,215, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243,245, 248, 250, 252, 254, 256, 258, 260, 262 and 264 can be created byintroducing one or more nucleotide substitutions, additions or deletionsinto the nucleotide sequence of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 38, 40, 42, 44, 46, 48, 50, 52,54, 56, 58, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87,88, 90, 91, 92, 94, 96, 98, 100, 102, 104, 106, 108, 109, 110, 112, 114,116, 118, 120, 122, 124, 125, 127, 129, 130, 132, 134, 135, 137, 139,141, 143, 145, 147, 149, 151, 153, 155, 156, 158, 160, 162, 164, 166,168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194,196, 198, 200, 202, 204, 206, 208, 210, 211, 213, 214, 216, 217, 218,220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246,247, 249, 251, 253, 255, 257, 259, 261, 263 and 265 such that one ormore amino acid substitutions, additions or deletions are introducedinto the encoded protein.

Mutations can be introduced into the nucleotide sequence of SEQ ID NO:1,3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 38,40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 59, 61, 63, 65, 67, 69, 71, 73,75, 77, 79, 81, 83, 85, 87, 88, 90, 91, 92, 94, 96, 98, 100, 102, 104,106, 108, 109, 110, 112, 114, 116, 118, 120, 122, 124, 125, 127, 129,130, 132, 134, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155,156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182,184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210,211, 213, 214, 216, 217, 218, 220, 222, 224, 226, 228, 230, 232, 234,236, 238, 240, 242, 244, 246, 247, 249, 251, 253, 255, 257, 259, 261,263 and 265 by standard techniques, such as site-directed mutagenesisand PCR-mediated mutagenesis. Preferably, conservative amino acidsubstitutions are made at one or more predicted non-essential amino acidresidues. A “conservative amino acid substitution” is one in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, a predicted nonessential amino acid residue in MYB68is replaced with another amino acid residue from the same side chainfamily. Alternatively, in another embodiment, mutations can beintroduced randomly along all or part of a MYB-subgroup14 or a MYB68coding sequence, such as by saturation mutagenesis, and the resultantmutants can be screened for biological activity to identify mutants thatretain activity and the desired phenotypes. Following mutagenesis of SEQID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 59, 61, 63, 65, 67, 69,71, 73, 75, 77, 79, 81, 83, 85, 87, 88, 90, 91, 92, 94, 96, 98, 100,102, 104, 106, 108, 109, 110, 112, 114, 116, 118, 120, 122, 124, 125,127, 129, 130, 132, 134, 135, 137, 139, 141, 143, 145, 147, 149, 151,153, 155, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178,180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206,208, 210, 211, 213, 214, 216, 217, 218, 220, 222, 224, 226, 228, 230,232, 234, 236, 238, 240, 242, 244, 246, 247, 249, 251, 253, 255, 257,259, 261, 263 and 265 the encoded protein can be expressed by anyrecombinant technology known in the art and the activity of the proteincan be determined.

Transformed Plants Cells and Transgenic Plants

The invention includes a protoplast, plants cell, plant tissue and plant(e.g., monocot or dicot) transformed with a MYB-subgroup14 nucleic acid,a vector containing a MYB-subgroup14 nucleic acid or an expressionvector containing a MYB-subgroup14 nucleic acid. As used herein, “plant”is meant to include not only a whole plant but also a portion thereof(i.e., cells, and tissues, including for example, leaves, stems, shoots,roots, flowers, fruits and seeds).

The plant can be any plant type including, for example, species from thegenera Arabidopsis, Brassica, Oryza, Zea, Sorghum, Gossypium, Triticum,Glycine, Pisum, Phaseolus, Lycopersicon, Trifolium, Cannabis, Cucurbita,Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trigonella,Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Raphanus, Sinapis,Atropa, Capsicum, Datura, Hyoscyamus, Nicotiana, Solanum, Petunia,Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus,Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum,Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Lolium, Avena,Hordeum, Secale, Picea, Caco, and Populus.

The invention also includes cells, tissues, including for example,leaves, stems, shoots, roots, flowers, fruits and seeds and the progenyderived from the transformed plant.

Numerous methods for introducing foreign genes into plants are known andcan be used to insert a gene into a plant host, including biological andphysical plant transformation protocols (See, for example, Miki et al.,(1993) “Procedure for Introducing Foreign DNA into Plants”, In: Methodsin Plant Molecular Biology and Biotechnology, Glick and Thompson, eds.,CRC Press, Inc., Boca Raton, pages 67-88; and Andrew Bent in, Clough S Jand Bent A F, (1998) “Floral dipping: a simplified method forAgrobacterium-mediated transformation of Arabidopsis thaliana”). Themethods chosen vary with the host plant, and include chemicaltransfection methods such as calcium phosphate, polyethylene glycol(PEG) transformation, microorganism-mediated gene transfer such asAgrobacterium (Horsch, et al., Science, 227: 1229-31 (1985)),electroporation, protoplast transformation, micro-injection, flowerdipping and biolistic bombardment.

Agrobacterium-Mediated Transformation

The most widely utilized method for introducing an expression vectorinto plants is based on the natural transformation system ofAgrobacterium tumefaciens and A. rhizogenes which are plant pathogenicbacteria which genetically transform plant cells. The Ti and Ri plasmidsof A. tumefaciens and A. rhizogenes, respectfully, carry genesresponsible for genetic transformation of plants (See, for example,Kado, Crit. Rev. Plant Sci., 10: 1-32 (1991)). Descriptions of theAgrobacterium vector systems and methods for Agrobacterium-mediated genetransfer are provided in Gruber et al., supra; and Moloney, et al, PlantCell Reports, 8: 238-242 (1989).

Transgenic Arabidopsis plants can be produced easily by the method ofdipping flowering plants into an Agrobacterium culture, based on themethod of Andrew Bent in, Clough S J and Bent A F, 1998. Floral dipping:a simplified method for Agrobacterium-mediated transformation ofArabidopsis thaliana. Wild type plants are grown until the plant hasboth developing flowers and open flowers. The plants are inverted for 1minute into a solution of Agrobacterium culture carrying the appropriategene construct. Plants are then left horizontal in a tray and keptcovered for two days to maintain humidity and then righted and bagged tocontinue growth and seed development. Mature seed is bulk harvested.

Direct Gene Transfer

A generally applicable method of plant transformation ismicroprojectile-mediated transformation, where DNA is carried on thesurface of microprojectiles measuring about 1 to 4 μm. The expressionvector is introduced into plant tissues with a biolistic device thataccelerates the microprojectiles to speeds of 300 to 600 m/s which issufficient to penetrate the plant cell walls and membranes. (Sanford, etal., Part. Sci. Technol., 5: 27-37 (1987); Sanford, Trends Biotech, 6:299-302 (1988); Sanford, Physiol. Plant, 79: 206-209 (1990); Klein, etal., Biotechnology, 10: 286-291 (1992)).

Plant transformation can also be achieved by the Aerosol Beam Injector(ABI) method described in U.S. Pat. No. 5,240,842, U.S. Pat. No.6,809,232. Aerosol beam technology is used to accelerate wet or dryparticles to speeds enabling the particles to penetrate living cellsAerosol beam technology employs the jet expansion of an inert gas as itpasses from a region of higher gas pressure to a region of lower gaspressure through a small orifice. The expanding gas accelerates aerosoldroplets, containing nucleic acid molecules to be introduced into a cellor tissue. The accelerated particles are positioned to impact apreferred target, for example a plant cell. The particles areconstructed as droplets of a sufficiently small size so that the cellsurvives the penetration. The transformed cell or tissue is grown toproduce a plant by standard techniques known to those in the applicableart.

Regeneration of Transformants

The development or regeneration of plants from either single plantprotoplasts or various explants is well known in the art (Weissbach andWeissbach, 1988). This regeneration and growth process typicallyincludes the steps of selection of transformed cells, culturing thoseindividualized cells through the usual stages of embryonic developmentthrough the rooted plantlet stage. Transgenic embryos and seeds aresimilarly regenerated. The resulting transgenic rooted shoots arethereafter planted in an appropriate plant growth medium such as soil.

The development or regeneration of plants containing the foreign,exogenous gene that encodes a polypeptide of interest introduced byAgrobacterium from leaf explants can be achieved by methods well knownin the art such as described (Horsch et al., 1985). In this procedure,transformants are cultured in the presence of a selection agent and in amedium that induces the regeneration of shoots in the plant strain beingtransformed as described (Fraley et al., 1983). In particular, U.S. Pat.No. 5,349,124 (specification incorporated herein by reference) detailsthe creation of genetically transformed lettuce cells and plantsresulting therefrom which express hybrid crystal proteins conferringinsecticidal activity against Lepidopteran larvae to such plants.

This procedure typically produces shoots within two to four months andthose shoots are then transferred to an appropriate root-inducing mediumcontaining the selective agent and an antibiotic to prevent bacterialgrowth. Shoots that rooted in the presence of the selective agent toform plantlets are then transplanted to soil or other media to allow theproduction of roots. These procedures vary depending upon the particularplant strain employed, such variations being well known in the art.

Preferably, the regenerated plants are self-pollinated to providehomozygous transgenic plants, or pollen obtained from the regeneratedplants is crossed to seed-grown plants of agronomically important,preferably inbred lines. Conversely, pollen from plants of thoseimportant lines is used to pollinate regenerated plants. A transgenicplant of the present invention containing a desired polypeptide iscultivated using methods well known to one skilled in the art.

A preferred transgenic plant is an independent segregant and cantransmit the MYB68 gene and its activity to its progeny. A morepreferred transgenic plant is homozygous for the gene, and transmitsthat gene to all offspring on sexual mating. Seed from a transgenicplant may be grown in the field or greenhouse, and resulting sexuallymature transgenic plants are self-pollinated to generate true breedingplants. The progeny from these plants become true breeding lines thatare evaluated for increased expression of the MYB68 transgene.

Method of Producing Transgenic Plants

Included in the invention are methods of producing a transgenic plant.The method includes introducing into one or more plant cells a compoundthat alters expression or activity of a MYB-subgroup14 in the plant togenerate a transgenic plant cell and regenerating a transgenic plantfrom the transgenic cell. The compound increases MYB-subgroup14expression or activity. The increased expression and or activity canadditionally be directed to occur ectopically or constitutively or in atissue specific manner. The compound can be, e.g., (i) a MYB-subgroup14polypeptide; (ii) a MYB-subgroup14 nucleic acid and analogs and homologsthereof; (iii) a nucleic acid that increases expression of aMYB-subgroup14 nucleic acid. A nucleic acid that increases expression ofa MYB-subgroup14 nucleic acid may include promoters or enhancerelements. The promoter is a heterologous promoter or a homologouspromoter. Additionally, the promoter is a constitutive or an induciblepromoter. Promoters include for example, organ specif promoter or tissuespecific promoter. Promoter suitable for directing gene expression areknow in the art and are described herein. Enhancer elements are known tothose skilled in the art. For example the enhancer element is a 35Senhancer element.

By increasing the expression of a MYB subgroup-14 polypeptide is meantthat the amount produced by the cell transformed with the nucleic acidconstruct is greater than a cell, e.g. control cell that is nottransformed with the nucleic acid construct. A control cell includes forexample a cell that endogenously expresses a MYB subgroup-14 polypeptidesuch as a plant root cell, alternatively a control cell is a nontransformed cell of the same cell-type as the transformed cell, be it aleaf cell a meristem cell or a flower or seed cell. An increase is a1-fold, 2-fold, 3 fold or greater increase. An increase of expression isalso meant to include expression of a MYB subgroup-14 polypeptide in acell that does not typically express a MYB subgroup-14 polypeptide.

The nucleic acid can be either endogenous or exogenous. Preferably, thecompound is a MYB-subgroup14 polypeptide or a MYB-subgroup14 nucleicacid encoding a MYB-subgroup14 polypeptide. For example the compoundcomprises the nucleic acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 38, 40, 42, 44, 46, 48,50, 52, 54, 56, 58, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83,85, 87, 88, 90, 91, 92, 94, 96, 98, 100, 102, 104, 106, 108, 109, 110,112, 114, 116, 118, 120, 122, 124, 125, 127, 129, 130, 132, 134, 135,137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 156, 158, 160, 162,164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190,192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 211, 213, 214, 216,217, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242,244, 246, 247, 249, 251, 253, 255, 257, 259, 261, 263 and 265.Preferably, the compound is a MYB-subgroup14 nucleic acid sequence froman endogenous source to the species being transformed. Alternatively,the compound is a MYB-subgroup14 nucleic acid sequence from an exogenoussource to the species being transformed.

Also included in the invention are methods of producing a transgenicplant. The method includes introducing into one or more plant cells acompound that alters a MYB-subgroup14 nucleic acid expression oractivity in the plant to generate a transgenic plant cell andregenerating a transgenic plant from the transgenic cell. The compoundincreases a MYB-subgroup14 sequence expression or activity. The compoundcan be, e.g., (i) a MYB-subgroup14 polypeptide; (ii) a MYB-subgroup14nucleic acid and analogs and homologs thereof; (iii) a nucleic acid thatincreases expression of a MYB-subgroup14 nucleic acid. A nucleic acidthat increases expression of a MYB-subgroup14 nucleic acid may includepromoters or enhancer elements. The nucleic acid can be eitherendogenous or exogenous. Preferably, the compound is a MYB-subgroup14polypeptide or a MYB-subgroup14 nucleic acid. For example the compoundcomprises the nucleic acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 38, 40, 42, 44, 46, 48,50, 52, 54, 56, 58, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83,85, 87, 88, 90, 91, 92, 94, 96, 98, 100, 102, 104, 106, 108, 109, 110,112, 114, 116, 118, 120, 122, 124, 125, 127, 129, 130, 132, 134, 135,137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 156, 158, 160, 162,164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190,192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 211, 213, 214, 216,217, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242,244, 246, 247, 249, 251, 253, 255, 257, 259, 261, 263 and 265.Preferably, the compound is a MYB-subgroup14 nucleic acid sequenceendogenous to the species being transformed. Alternatively, the compoundis a MYB-subgroup14 nucleic acid sequence exogenous to the species beingtransformed.

An exogenous MYB-subgroup14 sequence expressed in a host species neednot be identical to the endogenous MYB-subgroup14 sequence. For example,sequences of three Arabidopsis GAMYB-like genes were obtained on thebasis of sequence similarity to GAMYB genes from barley, rice, and L.temulentum. These three Arabidopsis genes were determined to encodetranscription factors (AtMYB33, AtMYB65, and AtMYB101) and couldsubstitute for a barley GAMYB and control alpha-amylase expression(Gocal et al. (2001) Plant Physiol. 127: 1682 1693).

Maize, petunia and Arabidopsis MYB transcription factors that regulateflavonoid biosynthesis are very genetically similar and affect the sametrait in their native species, therefore sequence and function of theseMYB transcription factors correlate with each other in these diversespecies (Borevitz et al. (2000) Plant Cell 12: 2383-2394).

Therefore an expressed MYB-subgroup14 need only be functionallyrecognized in the host cell. Expression of MYB-subgroup14 encodingnucleic acids in Arabidopsis provides the basis of a functionallyequivalent assay. For example expression of a MYB-subgroup14 from aBrassica, soybean, cotton or corn source in Arabidopsis and assessmentof the heat tolerance demonstrates functional equivalence and provides asound basis for prediction that the exogenous sequence is aMYB-subgroup14 gene and functions accordingly.

Disclosed herein is a description of expression of MYB-subgroup14sequences from Arabidopsis, Brassica and soybean that have beendemonstrated to be functional in Arabidopsis in that the resultingplants have increased heat tolerance as indicated by reduced flowerabortion and increased seed set under heat stress conditions duringflowering.

In various aspects the transgenic plant has an altered phenotype ascompared to a wild type plant (i.e., untransformed). By alteredphenotype is meant that the plant has a one or more characteristic thatis different from the wild type plant. For example, when the transgenicplant has been contacted with a compound that increases the expressionor activity of a MYB-subgroup14 nucleic acid, the plant has a phenotypesuch as increased heat tolerance as compared to a wild type plant andmanifests this trait in phenotypes such as decreased flower abortion,increased seed set and development, increased yield protection andprotection of pollen development and protection of meristems,particularly flower meristems, from heat damage, drought tolerance andsalt tolerance for example. Plants with a reduced flower abortion have a5, 10, 20, 25, 30% or more increase in seed yield as compared to acontrol plant.

The plant can be any plant type including, for example, species from thegenera Arabidopsis, Brassica, Oryza, Zea, Sorghum, Gossypium, Triticum,Glycine, Pisum, Phaseolus, Lycopersicon, Trifolium, Cannabis, Cucurbita,Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trigonella,Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Raphanus, Sinapis,Atropa, Capsicum, Datura, Hyoscyamus, Nicotiana, Solanum, Petunia,Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus,Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum,Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Lolium, Avena,Hordeum, Secale, Picea, Caco, and Populus.

Method of Identifying a Heat Stress Tolerant Plant

Also included in the invention is a method of identifying a heat stresstolerant plant. The plants identified by these methods have reducedflower abortion and increased yield as compared to a control plant. Heatstress tolerant plants are identified by exposing a population offlowering plants to a heat stress treatment and selecting a plant fromthe population of plants that has reduced flower abortion. Heat stresstreatment includes for example exposing the plant to a temperature thatis hot enough for a sufficient amount of time such that damage to plantfunctions or development results. By reduced flower abortion is meantthat a plant does not loss as many flowers, due to flower abortion, orhas a greater seed yield compared to another plant that is exposed to asimilar level of heat stress. Plants with a reduced flower abortion havea 5, 10, 20, 25, 30% or more increase in seed yield as compared to acontrol plant.

Examples

The invention will be further illustrated in the following non-limitingexamples.

Example 1: Identification of Heat Tolerant Mutant

Arabidopsis thaliana var. Columbia was transformed with pSKI15 vectorcontaining a 4×35S enhancer sequence (Wiegel et al., 2000). A T3population of Arabidopsis seed was obtained from ABRC and used toproduce a T4-generation that was used in genetic screen experiments. TheArabidopsis h138 mutant was identified as having reduced or no flowerabortion, relative to a wild type control, when exposed to a heat stressduring flowering of about 45° C. for about 30 to 60 minutes. Initialisolates were retested by having flowering plants subjected to a 1 hourtemperature ramp-up from 22° C. to 45° C. followed by a 2 hour heatstress of 45° C., flower production, seed set and seed development wasmonitored and heat tolerant lines selected.

Example 2: Identification of the Heat Tolerant MYB68 Gene

Genome walking to localize the T-DNA activation tag insertion wasperformed as follows. Genomic DNA was purified by phenol:chloroformextraction using 10-day-old seedlings of mutant h138. The isolated DNAwas subsequently digested by the restriction enzymes such as EcoRV,PvuII, NruI, or StuI to generate DNA fragments with blunt ends. Theresulting fragments from each digestion were ligated to an adaptor thatwas formed by the annealing of two oligos: Adaptor 1 and Adaptor 2. Theaddition of the adaptor to the DNA fragments enables PCR amplificationusing primers specific to the adaptor and a T-DNA insertion site.

Two rounds of PCR were used to generate DNA fragments for furthersequencing analysis. Primer HeatL1 (SEQ) that is specific to the T-DNAleft border, and primer CAP1 (SEQ) that is specific to the adaptor, wereused for the 1^(st) PCR. The resulted PCR products were diluted 50 foldsto serve as templates for 2^(nd) PCR. A confirmed DNA fragment was thenamplified by two nested primers HeatL2 (SEQ) and CAP2 (SEQ). PCRprograms TOUCH1 (6 cycles of 94° C., 25 sec; 72° C., 7 min; 32 cycles of94° C., 25 sec; 67° C., 7 min and 1 cycle of 67° C., 10 min) and TOUCH2(4 cycles of 94° C., 25 sec; 72° C., 7 min; 20 cycles of 94° C., 25 sec;67° C., 7 min and 1 cycle of 67° C., 10 min) were used for the tworounds of PCR. All PCR was carried out using Ex-Taq as DNA polymeraseand a Biometra® thermocycler. The PCR products were sequenced, and theflanking genomic sequences identified. The 4×35S enhancers were insertedinto an intergenic region that is 5 kb down stream of 3′ end of genomicAtMYB68 (AT5G65790) on chromosome 5. Northern analysis and real-time PCRshowed that the expression of MYB68 in h138 was induced to more than 2fold relative to wild type.

Example 3: Physiological Characterization of the h138 Mutant (myb68)

Plants were assessed for heat tolerance during flowering and scoredbased on the number of aborted flowers or pods and final seed yield.Plants were grown in controlled environment chambers where optimalgrowth conditions were 16 hr light 200 uE and 8 hr dark, 22° C. and 70%relative humidity. Three groups of plants were used in the experimentaldesign; 1) A control group grown under optimal conditions; 2) a 3-hourheat treatment group and; 4) a 4-hour heat treatment group. Heattreatment was performed 6 days after first open flower and thetemperature was ramped from 22° C. to 44° C. over a 1-hour period. Eachgroup of plants contained the myb68 mutant and its wild type control(myb68-null) with 10 replicate pots per entry per treatment with eachpot containing 5 plants. Plants were assessed for flower abortion a weekfollowing the heat stress treatments then left to grow under optimalconditions until maturity. Final seed yield per pot was determined forall 3 groups of plants.

Following heat stress the seed yield of myb68 was lower than that of themyb68-null control in both the 3-hour (25%) and 4-hour (17%) stresstreatments however the difference was only statistically significant forthe 3-hour treatment. The 3-hour treatment resulted in 32% fewer abortedpods relative to myb68-null and the final seed yield was increased by16% relative to myb68 plants grown in optimal conditions. The 4-hourtreatment also resulted in a 16% increase in seed yield relative tooptimally grown myb68 plants. In contrast, the myb68-null showed 15% and23% reductions in seed yield relative to optimally grown plants. Theoverall yield protection provided by the myb68 mutation was 31% and 39%,relative to the wild-type. Additional experiments have shown results ofyield protection ranging from 5% to 44% depending on the experimentalconditions.

Example 4: Constructs Useful for Expression of MYB-Subgroup14 SequencesIncluding MYB68

According to the methods described below, expression vector constructscan be produced using appropriate promoters and a MYB gene of theinvention. For example any of the gene sequences described by the SEQ IDNO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 59, 61, 63, 65, 67, 69,71, 73, 75, 77, 79, 81, 83, 85, 87, 88, 90, 91, 92, 94, 96, 98, 100,102, 104, 106, 108, 109, 110, 112, 114, 116, 118, 120, 122, 124, 125,127, 129, 130, 132, 134, 135, 137, 139, 141, 143, 145, 147, 149, 151,153, 155, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178,180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206,208, 210, 211, 213, 214, 216, 217, 218, 220, 222, 224, 226, 228, 230,232, 234, 236, 238, 240, 242, 244, 246, 247, 249, 251, 253, 255, 257,259, 261, 263 and 265. Such vector constructs are useful to produce aMYB68 gene, operably linked to a sequence that functions as a promoterin a plant cell and to operably express said gene and protein encoded bythe gene.

Vectors to over-express MYB68 under regulatory control of eitherconstitutive or conditional promoters may be constructed, as describedbelow. The sequence encoding a MYB68 open reading frame has beenoperably linked to the promoter sequences of the 35S CaMV constitutivepromoter, the P18.2 or P81.1 heat inducible promoters and its endogenousPMYB68 promoter. Additionally the genomic sequence of MYB68 has beencloned behind the 35S CaMV constitutive promoter in a pEGAD vectorbackbone.

35S-Genonic AtMYB68 (in pEGAD Vector (35S-gAtMYB68)

A 1.4 kb of MYB68 genomic DNA including 83 bps of 3′ UTR was amplifiedby PCR using primers: MYB68FW-BamH3(5′-AAAGGATCCATGGGAAGAGCACCGTGTTG-3′) (SEQ ID NO:300) and MYB68RV-BamH4(5′-AAAGGATCCCCACTCCCTAAAGACACAGATTT-3′) (SEQ ID NO:301), andsubsequently digested with BamHI. The resulting DNA fragment was ligatedinto pBluescript II SK (+/−), and then subcloned into pEGAD at the samesite to obtain 35S-gemonicAtMYB68 (35S-gAtMYB68) in pEGAD.

35S-AtMYB68, 35S-AtMYB84, 35S-AtMYB36, 35S-AtMYB37, 35S-AtMYB38,35S-AtMYB87

AtMYB84 (At3g49690), AtMYB36 (At5g57620), AtMYB37 (At5g23000), AtMYB38(At2g36890) and AtMYB87 (At4g37780) are classified as members of theMYB-subgroup14 family along with AtMYB68 (Stracke et al., 2001),therefore it is possible that their functions are redundant. These MYBgenes are over-expressed in Arabidopsis to test their functionality asan AtMYB68 orthologue with respect to heat tolerance. The cDNA sequencesare amplified by RT-PCR, and cloned into pBI121 without GUS to generateconstructs of 35S-AtMYB84, 35S-AtMYB36, 35S-AtMYB37, 35S-AtMYB38 and35S-AtMYB87.

A 1.1 kb of AtMYB68 cDNA was produced by RT-PCR using primers HG2F(5′-AAATCTAGAATGGGAAGAGCACCGTGTT-3′) (SEQ ID NO:302) and HG2R(5′-AAAGGATCCTTACACATGATTTGGCGCAT-3′) (SEQ ID NO:303), and digested withXbaI and BamHI. The resulting DNA fragment was cloned into pBluescriptII SK (+/−), and then into pBI121 without GUS to generate 35S-MYB68.pBI121 without GUS was obtained by SmaI and EcolcR1 double digestion andfollowed by self-ligation of the remaining vector.

The coding sequence of AtMYB84 (933 bp, AtMYB84, At3g49690) wasamplified by RT-PCR using forward primer 690M84-Xba-FW containing anXbaI site (5′-acgt TCTAGA ATG GGA AGA GCA CCG TGT TG-3′) (SEQ ID NO:273)and reverse primer 690M84-Bam-Re containing a BamHI site (5′-atcg GGATCCTTA AAA AAA TTG CTT TGA ATC AGA ATA-3′) (SEQ ID NO:274). The PCR productwas cloned at the XbaI-BamHI sites in pBI121, generating construct of35S-AtMYB84.

The coding sequence of AtMYB36 (1002 bp, AtMYB36, At5g57620) wasamplified by RT-PCR using forward primer M36-Xb-FW containing an XbaIsite (5′-actg TCTAGA ATG GGA AGA GCT CCA TGC TG-3′) (SEQ ID NO:304) andreverse primer M36-Bm-Re containing a BamHI site (5′-cagt GGATCC TTA AACACT GTG GTA GCT CAT C-3′) (SEQ ID NO:305). The PCR product was cloned atthe XbaI-BamHI sites in pBI121, generating construct of 35S-AtMYB36.

The coding sequence of AtMYB37 (990 bp, AtMYB37, At5g23000) wasamplified by RT-PCR using forward primer AM37-Xb-FW containing an XbaIsite (5′-actg TCTAGA ATG GGA AGA GCT CCG TGT TG-3′) (SEQ ID NO:306) andreverse primer AM37-Bm-Re containing a BamH I site (5′-acgt GGATC CTAGGA GTA GAA ATA GGG CAA G-3′) (SEQ ID NO:307). The PCR product wascloned at the XbaI-BamHI sites in pBI121, generating construct of35S-AtMYB37.

The coding sequence of AtMYB38 (897 bp, AtMYB38, At2g36890) wasamplified by RT-PCR using forward primer AM38-Xb-FW containing an XbaIsite (5′-actg TCTAGA ATG GGT AGG GCT CCA TGT TGT-3′) (SEQ ID NO:308) andreverse primer AM38-Bm-Re containing a BamH I site (5′-acgt GGATCC TCAGTA GTA CAA CAT GAA CTT ATC-3′) (SEQ ID NO:309). The PCR product wascloned at the XbaI-BamHI sites in pBI121, generating construct of35S-AtMYB38.

The coding sequence of AtMYB87 (918 bp, AtMYB87, At4g37780) will beamplified by RT-PCR using forward primer M87-Xb-FW containing an XbaIsite (5′-aaaa TCTAGA ATG GGA AGA GCA CCG TGC-5′) (SEQ ID NO:310) andreverse primer M87-Bg-Re containing a Bgl2 site (5′-aaaa AGATCT CTA CTCATT ATC GTA TAG AGG-3′) (SEQ ID NO:311). The PCR product will be clonedat the XbaI-BamHI sites in pBI121, generating construct of 35S-AtMYB87.

P18.2-MYB68, and P81.1-MYB68

The construction involved 4-steps; 1) a 869 bp of Hsp18.2 promoter, anda 406 bp of Hsp81.1 promoter were amplified by PCR using primer sets:HP1F (SEQ ID NO: 277) and HP1R (SEQ ID NO:278), and HP2F (SEQ ID NO:279)and HP2R (SEQ ID NO:280), respectively, and digested with SalI and XbaI.The resulting DNA fragments were cloned into pBI101 at the same sites togenerate the new vectors: P18.2pBI101 and P81.1pBI101; 2) a MCS2-oligo(including restriction sites of XbaI, HpaI, AgeI, KpnI, XhoI, ScaI,SpeI, SalI, BamHI and SmaI) was cloned into the new vectors at XbaI andSmaI sites. The resulting vectors were named P18.2pBI101MCS andP81.1pBI101MCS; 3) the GUS gene was removed by SmaI and EcolcR1 doubledigestion and followed by self-ligation of the remaining vector to givevectors P18.2pBI101MCS without GUS and P81.1pBI101MCS without GUS; 4)the 1.1 kb of MYB68 cDNA fragment was ligated into the two newer vectorsat XbaI and BamHI sites to complete the construction of P18.2pBI101MCSwithout GUS for P18.2-MYB68, and P81.1MCSpBI121 without GUS forP81.1-MYB68.

pHSP81.1-AtMYB68

The coding sequence of AtMYB68 was isolated by restriction digestionwith XbaI and BamHI from plasmid pHSP18.2-AtMYB68, and cloned at theXbaI-BamHI sites in pHSP81.1.

pHPR-AtMYB68

The promoter sequence (−1 to −506 bp, relative to ATG start codon) ofthe Arabidopsis hydroxy pyruvate reductase gene (HPR, At1g68010) wasamplified by PCR from Arabidopsis genomic DNA using a forward primercontaining a Sal I site (HPR-Sal-FW, acgt gtcgac GAAGCAGCAGAAGCCTTGAT)(SEQ ID NO:312) and a reverse primer containing an Xba I site(HPR-Xb-R2, acgt tctaga GGT AGA GAA AAG AGA aag cct c) (SEQ ID NO:313).The digested fragment was cloned into the vector pHSP81.1-AtMYB68 thatwas pre-digested with SalI and XbaI to remove the HSP81.1 promoter. Thisgenerates a recombinant plasmid with the HPR promoter placed in front ofAtMYB68.

PMYB68-AtMYB68

The AtMYB68 promoter (−1 through −1034 with respect to the MYB68 ATGstart codon) was amplified by PCR using primers: Pm68-H3-FW (SEQ IDNO:275) and Pm68-Av-Xh-Re (SEQ ID NO:276), and digested by restrictionenzymes: HindIII and XhoI. The resulting promoter fragment was clonedinto P81.1MCSpBI121 without GUS at the same sites, replacing the Hsp81.1promoter. This vector is then named PMYB68pBI121, and used for furthercloning of AtMYB68 cDNA (1.1 kb) at Avr II and BamHI sites to obtainPMYB68-AtMYB68. The AvrII-BamHI fragment of MYB cDNA was recovered fromthe plasmid 18.2-MYB68.

pM68-AtMYB84

The coding sequence of AtMYB84 (933 bp, AtMYB84, At3g49690) wasamplified by RT-PCR using forward primer 690M84-Xba-FW containing anXbaI site (5′-acgt TCTAGA ATG GGA AGA GCA CCG TGT TG-3′) (SEQ ID NO:273)and reverse primer 690M84-Bam-Re containing a BamHI site (5′-atcg GGATCCTTA AAA AAA TTG CTT TGA ATC AGA ATA-3′) (SEQ ID NO:274). The PCR productwas cloned at the AvRII-BamHI sites in pB-Pm68, generating construct ofAtMYB84 under control of the AtMYB68 promoter.

pM68-AtMYB36

The coding sequence of AtMYB36 (1002 bp, At5g57620) was amplified fromRNA isolated from young Arabidopsis seedlings (leaves and roots) byRT-PCR using forward primer M36-Xb-FW containing an XbaI site (5′-actgTCTAGA ATG GGA AGA GCT CCA TGC TG-3′) (SEQ ID NO:305) and reverse primerM36-Bm-Re containing a BamHI site (5′-cagt GGATCC TTA AAC ACT GTG GTAGCT CAT C-3′) (SEQ ID NO:306). The PCR product was cloned at the Avr IIand BamHI sites in pBI-Pm68 described above. This generated a constructof AtMYB36 under control of the AtMYB68 promoter.

35S-OsMYB36

Rice MYB36 cDNA homolog: The coding sequence (966 bp) of a rice MYB36gene (SEQ ID NO:9), encoding a protein identified as SEQ ID NO:10 wasamplified by RT-PCR from rice root RNA using forward primer rM-Xb-FW2containing an XbaI site (5′-acgt TCTAGA ATG GGG AGA GCG CCG TGC TG-3′)(SEQ ID NO:314) and reverse primer rM-Bm-Re2 containing a BamH I site(5′-tgca GGATCC CTA CTG CAT CCC GAG GTC AG CT-3′) (SEQ ID NO:315). ThePCR product was cloned at the XbaI-BamHI sites in pBI121, generatingconstruct 35S-Os MYB36.

35S-gOs MYB36

Rice MYB genomic homolog clone: Using the same primers described aboveforward primer rM-Xb-FW2 (SEQ ID NO:314) and reverse primer rM-Bm-Re2(SEQ ID NO:315), the genomic sequence of the rice MYB36 gene (SEQ IDNO:265) was amplified (1259 bp). The PCR product was cloned at theXbaI-BamHI sites in pBI121, generating construct 35S-gOsMYB36.

35S-GmMYB84

The soybean MYB161 is a homolog of Arabidopsis MYB84. Herein the term‘soybean MYB84’ is used interchangeably with Soybean MYB161. The 1068 bpcoding sequence of a soybean MYB161 was cloned by RT-PCR from soybeanroot RNA using forward primer soybM-Xba-FW2 containing an XbaI site(5′-acgt TCTAGA ATG GGG AGG GCA CCT TGC T-3′) (SEQ ID NO:316) andreverse primer soybM-Bm-Re containing a BamHI site (5′-acgt GGATC CTATTG CGC CCC CGG GTA G-3′) (SEQ ID NO:317). The PCR product was cloned atthe XbaI-BamHI sites in pBI121, generating construct 35S-GmMYB84.

35S-ZmMYB36

The corn MYB36 cDNA (SEQ ID NO:261) was amplified by PCR using primers:ZmYYBFW-XbaI (5′-aaatctagaATGGGGAGAGCTCCGTGCTGCGACA-3′) (SEQ ID NO:318)and ZmMYBRV-BamHI2 (5′-aaaggatccCTACTTCATCCCAAGGTTTCCTGGC-3′) (SEQ IDNO:319). The DNA fragment was digested by XbaI and BamHI andsubsequently ligated to the same sites at pBluescript II SK (+), andthen subcloned into the same sites of pBI121 replacing GUS.

35S-GhMYB68 or 35SS-CotMYB68

The cotton MYB68 cDNA was amplified by PCR using primers: CotM-Xb-Fw(5′-acgt TCTAGA ATG GGG AGA GCT CCT TGT TG-3′) (SEQ ID NO:320) andCotM-Bm-Re (5′-acgt GGATCC CTA TTG CGC TCC TCC TGG G-3′) (SEQ IDNO:321). The DNA fragment was digested by XbaI and BamHI. It was ligatedto the same sites at pBluescript II SK (+), and then subcloned into thesame sites in pBI121 replacing GUS.

35S-BnMYB68r

The canola root MYB cDNA was amplified by PCR using primers:Bn68root-FW-XbaI (5′-aaatctagaATGGGAAGAGCACCGTGTTGTGATAAGGCC-3′) (SEQ IDNO:322) and Bn68root-RV-BamHI(5′-aaaggatccTTACACATTATTTGGCCCATTGAAGTATCTTGC-3′) (SEQ ID NO:323). TheDNA fragment was digested by XbaI and BamHI. It was ligated to the samesites at pBluescript II SK (+). The same fragment was then subcloned tothe same sites in pBI121 replacing GUS.

35S-BnMYB68b

The canola bud MYB cDNA was amplified by PCR using primers:Bn68Bud-FW-XbaI (5′-aaatctagaATGGGAAGAGCACCGTGTTGTGACAAGGCT-3′) (SEQ IDNO:324) and Bn68Bud-RV-BamHI(5′-aaaggatccTTACAAATGATTTGCCCCATTGAAGTAACTTGC-3′) (SEQ ID NO:325). TheDNA fragment was digested by XbaI and BamHI. It was ligated to the samesites at pBluescript II SK (+). The same fragment was then subcloned tothe same sites in pBI121 replacing GUS.

Table 2 below describes oligonucleotide primers used to make the vectorconstructs described above, and additional primers useful for cloningAtMYB homologues.

TABLE 2Oligonucleotide primers synthesized for cloning AtMYB68 homologuesSEQ ID Restriction NO Primer name site Sequence (5′-3′) Remark 271790M68-Xba- XbaI ACGT TCTAGA ATG GGA AGA AtMYB68 FW GCA CCG TGT TG(at5g65790) 272 790M68-Bam- BamHI ATCG GGATCC TTA CAC ATG ATT AtMYB68 ReTGG CGC ATT G (at5g65790) 273 690M84-Xba- Xba I ACGT TCTAGA ATG GGA AGAAtMYB84 FW GCA CCG TGT TG (at3g49690) 274 690M84-Bam- Bam HIATCG GGATCC TTA AAA AAA TTG AtMYB84 Re CTT TGA ATC AGA ATA (at3g49690)275 Pm68-H3-FW Hind III ACGT AAGCTT TCG TAA AAT CTC AtMYB68 TCA TGPromoter 276 Pm68-Av-Xh- Avr II and GTCA CTCGAG CCTAGG TTT CTT AtMYB68Re Xho I GAT TCT TGA TTC TTG ATC Promoter 277 HP1fAAAGTCGACGCATCTTTACAATGT AAAGCTTTTCT 278 HP1R AAATCTAGATGTTCGTTGCTTTTCGGG 279 HP2F AAAGTCGACAGAAGACAAATGAG AGTTGGTTTATATTT 280 HP2RAAATCTAGACGCAACGAACTTTG ATTCAA 281 BnMYB68FW2 ATGGGAAGAGCACCGTGTTGTGACanola MYB68 TAAGGCC (AC189266.1) 282 BnMYB68RV2TTAATTTGGCGCATTGAAGTAACT Canola MYB68 TGCATCTTCGG (AC189266.1) 283rM-Xb-FW Xba I ACGT TCTAGA ATG GGG AGA Rice MYB GCG CCG TGC (AAT85046)284 rM-Bm-Re BamH I TGCA GGATC CTA CTG CAT CCC Rice MYB GAG GTC AG(AAT85046) 285 cotM-Xb-FW Xba I ACGT TCTAGA ATG GGG AGA Cotton MYBGCT CCT TGT TG (TC34239) 286 cotM-Bm-Re BamH IACGT GGATCC CTA TTG CGC TCC TCC TGG G 287 soybM-Xba- XbaIACGT TCTAGA ATG GGG AGG Soybean MYB FW2 GCA CCT TGC T (ABH02906) 288cornM-Xba- XbaI I ACGT TCTAGA ATG GGG AGA Corn MYB FW2 GCT CCG TGC T(TC370133) 289 wheatM-Xba- XbaI ACTG TCTAGA ATG GGG AGG Wheat MYB FWGCG CCG TGC (BQ483726) 290 MedtM-Xba- XbaI ACTG TCTAGA ATG GGA AGAM. truncatula FW GCT CCT TGC TGT MYB (TC97441) 291 sorgM-Xb-FW XbaIACGTTCTAGA ATGGGGAGAG sorghum MYB CTCCGTGCT (AAL84760) 292 toM-Xb-FWXbaI ACTGTCTAGAATGGGAAGAGCTC Tomato blind CATGTTGT (AAL69334) 293toM-Bm-Re BamtII GACT GGATCC TTA GTA ATA AAA CAT CCC TAT CTC A 294popM-Xb-FW Xba I ACGT TCTAGA ATG GGG AGA Poplar MYB GCT CCT TGC TG(TC54478) 295 popM-Bm-Re BamtII GACT GGATCC TCA TTG TGG CCC Poplar MYBAAA GAA GCT (TC54478) 296 HSP18.2 HP1F AAAGTCGACGCATCTTTACAATGTAAAGCTTTTCT 297 HSP18.2 HP1R AAATCTAGATGTTCGTTGCTTTTC GGG 298 HSP81.1HP2F AAAGTCGACAGAAGACAAATGAG AGTTGGTTTATATTT 299 HSP81.1 HP2RAAATCTAGACGCAACGAACTTTG ATTCAA Note: 1. FW: Forward primer with genespecific sequence starting from the ATG start codon. 2. Re: Reverseprimer with gene specific sequence starting at the stop codon. 3.Restriction sites at the 5′ end are underlined.

The expression vector constructs of the invention can be introduced intoArabidopsis, the plant of origin or any species of choice. For examplean Arabidopsis MYB gene may be over expressed in a Brassica species oralternatively a soybean, maize, rice or cotton species.

Example 5: Amino Acid Sequence Analysis of MYB68

The tables below provide a comparison of amino acid sequences from theMYB gene family across different plant species, under different settingsfor multiple sequence alignment and amino acid sequence analysis. Note:Different MYB naming and numbering systems are used in the literatureand databases for different plant species. In the tables below, (*)indicates the predicted ORF genomic DNA sequence was edited according topeptide sequence alignments to generate a putative coding sequencesequence. The (P) designation indicates a partial sequence. Sequencehomology and multiple sequence alignments were compared by ClustalW.

TABLE 3 Protein Multi-alignment scores to Sequence size AtMYBs (%,Clustal) Species Name file (a.a.) MYB68 MYB84 MYB36 AtMYB68 100 AtMYB84At3g49690 310 64 100 SEQ ID NO: 4 AtMYB36 At5g57650 333 35 39 100 SEQ IDNO: 6 Canola AC189266 364 (*) 88 59 33 SEQ ID NO: 8 Rice Rice MYB 360 3436 s8137 Rice MYB AAT85046 321 39 40 39 s3656 SEQ ID NO: 10 SoybeanSoybean ABH02831 259 21 20 MYB84 Soybean ABH02839 317 22 20 MYB84Soybean ABH02906 198 (P) 63 65 58 MYB161 SEQ ID NO: 14 Soybean ABH02912209 (P) 58 56 MYB71 Corn TC32080 361 33 38 TC32080 131 79 80 ZmMYB-AF099429 43 (P) 86 86 IP30 ZmMYB- TC370133 131 (P) 82 83 83 IP30 SEQ IDNO: 18 ZmMYB- AF099383 43 (P) 81 81 HX43 Corn CAJ42201 226 33 35 MYB8Corn CAJ42202 275 26 26 MYB31 Corn P20025 255 30 30 MYB38 Cotton CottonTC34239 356 40 41 34 MYB SEQ ID NO: 12 Cotton AAK19616 309 32 27 GHMYB25Cotton AAK19619 264 28 25 GHMYB9 Cotton AAZ83352 307 31 29 GHMYB30Sorghum Sorghum AAL90639 87 (P) 60 62 MYB68 Sorghum AAQ54875 157 (P) 6970 MYB86 Sorghum AAL84760 157 (P) 69 70 75 MYB20 SEQ ID NO: 20 SorghumAAL84761 203 46 40 MYB34 Sbi_042749 318 38 37 203 55 53 157 63 63Medicago ABE78637 336 28 31 truncatula ABE90877 319 24 24 TC97441 178(P) 70 70 66 SEQ ID NO: 26 Tomato Blind AAL69334 315 39 SEQ ID NO: 28EST467561 237 51 51 SEQ ID NO: 30 TC182203 185 (P) 60 54 64 AAL69334 18562 58 62 EST467561 185 61 58 59 Wheat BQ483726 175 (P) 66 65 70 SEQ IDNO: 22 Poplar TC54478 345 39 44 38 SEQ ID NO: 24

TABLE 4 ATMYB68 Homologues from Different Crops/Species ProteinMulti-alignment scores to AtMYBs Sequence size (%, ClustalW) Name file(a.a.) AtMYB68 AtMYB84 AtMYB36 AtMYB68 At5g65790 374 100 SEQ ID NO: 2AtMYB84 At3g49690 310 64 100 AtMYB36 At5g57620 333 35 39 100 CanolaAC189266 364 ((*)) 88 59 33 Soybean ABH02906 198 (P) 63 65 58 CottonTC34239 356 40 41 34 Tomato Blind 315 39 AAL69334 Medicago TC97441 178(P) 70 70 66 truncatula Rice AAT85046 321 39 40 38 Corn TC370133 131 (P)82 83 83 Wheat BQ483726 175 (P) 66 65 70 Sorghum AAL84760 157 (P) 69 7075 Poplar TC54478 345 39 44 38

In Table 4 above, sequence homology and multiple sequence alignmentswere compared by ClustalW at http://www.ebi.ac.uk/clustalw/ with thefollowing default settings:

Matrix: Gonnet 250

GAP OPEN: 10.0

END GAPS: −1

GAP EXTENSION: 0.2

GAP DISTANCES: 4

TABLE 5 ATMYB68 Homologues from Different Crops/Species Sequencehomology Protein size (%) to AtMYB68 Species Sequence file (a.a.)Protein DNA AtMYB68 At5g65790 374 100 100 SEQ ID NO: 2 AtMYB84 At3g49690310 64 68 AtMYB36 At5g57620 333 37 29 Canola AC189266 364 ((*)) 88 90Soybean ABH02906 198 (P) 63 53 Cotton TC34239 356 40 30 Tomato Blind 31539 28 (AAL69334) Medicago TC97441 178 (P) 70 58 truncatula Rice AAT85046321 39 28 Corn TC370133 131 (P) 82 67 Wheat BQ483726 175 (P) 66 53Sorghum AAL84760 157 (P) 69 59 Poplar TC54478 345 39 30

In Table 5 above, sequence homology and multiple sequence alignmentswere compared by ClustalW at http://www.ebi.ac.uk/clustalw/ with thefollowing default settings:

Matrix: Protein: Gonnet 250

GAP OPEN: DNA: 15.0 Protein: 10.0

END GAPS: −1

GAP EXTENSION: DNA: 6.66 Protein: 0.2

GAP DISTANCES: 4

Example 6: Physiological Characterization of the 35S-MYB68 ExpressionLines

Within a population of transgenic lines a gradation of expression levelsand physiological response will exist. In part, the gradation ofvariation is a result of the site of integration of the gene constructand the local environment for gene expression at that locus. Therefore,lines must be screened and evaluated in order to select the bestperforming lines. This process is one of routine to one skilled in theart.

Homozygous lines expressing a 35S-MYB68 expression construct have beenevaluated in a heat and flower abortion experiment. The experimental setup included 8 replicate plants per line with 1 plant per 3″ pot andgrown under optimal conditions in a controlled environment chamber (18hr light at 200 uE, 6 hr dark, 22° C., 70% relative humidity). Threedays after the appearance of the first flower, plants were exposed to aheat shock of 1 hour at 42° C. and returned to optimal conditions for afurther 7 days. Plants were assessed for flower abortion on the mainstem. Lines were identified that demonstrated reduced flower abortionrates from 34% to 60% relative to wild type controls.

Seed yields and yield protection, expressed as a percent relative towild type, was determined for nine independent 35S-MYB68 transgeniclines. The experimental set up included 3 plants per 3″ pot which weregrown as above with 22 replicates per line. Three days after theappearance of the first flower, 12 replicates per line were exposed to a3-hour heat stress at 45° C. with a 1 hour ramp up from 22° C. Threedays later the heat stress was applied again. The remaining 10replicates the plants per line were maintained under optimal conditionsthroughout their life cycle. The final seed yield was determined for allthe plants. Wild type plants showed a 40% reduction in yield due to theapplied heat stress whereas transgenic lines, while still having areduced yield due to heat stress the reduction was less severe resultingin a 10% to 12% yield protection.

Selected lines were re-evaluated and stressed as follows. Plants wereexposed to a 1-hour ramp up period from 22° C. to 45° C. and a heatstress of 45° C. for 1.5 to 1.8 hours was maintained. Heat stress wasapplied daily for five consecutive days followed by a five day recoveryperiod and then a sixth heat treatment. The heat treatment resulted in11% reduction in yield in WT plants and in four of the transgenic linesa yield increase was observed ranging from 2% to 17%. As shown in Table6, six transgenic lines (68, 80, 93, 73, 83, 30) showed yield protectionrelative to WT following the heat stress. This protection ranged from 3to 31%. Two of these transgenic lines (30 and 73) have been shown tohave yield protection in the previous experiment and two lines (93 and83) showed reduction in flower abortion.

TABLE 6 Yield protection Line Heat seed yield relative to WT 68 0.81631% 80 0.714 26% 93 0.781 17% 73 0.820 14% 83 0.719  5% 30 0.801  3% WT0.784 — myb68 0.577 15% WT (myb68- 0.779 — null)

Characterization of Arabidopsis Lines Expressing the PMYB68-AtMYB68Construct

Fourteen homozygous transgenic lines at T3 were evaluated in a flowerabortion experiment. Plants (fourteen replicates per entry) were grownunder optimal conditions (18 hr light, 200 uE, 22 C, 60% RH) in 2.25″pots until three days from first open flower. At that point plants wereexposed to 1 hr treatment at 43 C. Following the heat treatment plantswere returned to optimal conditions for 7 more days. The impact of heattreatment on the pod formation was assessed at that point by countingthe aborted and damaged (short) pods in the region that was exposed tothe heat stress. Six transgenic lines showed a reduction in the totalnumber of damaged pods as compared to controls (wild type-Colombia andthe null-n11-8) (see Table 7 below). The best line showed only 70% ofthe heat damage as the control. Two of the transgenic lines examinedhere also showed reduced flower abortion at the T2 stage of screening.The myb68 mutant, included as a positive control also showed a reductionin the total number of damaged pods as compared to its segregating nullcontrol (myb68-null).

Example 7: Physiological Assessment of Transgenic Plants ExpressingMYB68 Arabidopsis Myb68 Mutant Shows Drought Tolerance

Five plants per 3″ pot with 6 replicates per entry were grown underoptimal conditions of 16 hr light (180 uE), 22 C, 70% RH in a growthchamber until first open flower. A drought treatment was startedequalizing the amount of water in all pots and cessation of watering.Water loss was measured daily by weighing the pots. Soil water content(SWC) was calculated as a % of initial. Plants were harvested on days 0,2 and 4 of the drought treatment. Water lost relative to shoot drybiomass of the plants was calculated. The myb68 plants show trendstowards greater SWC than that of control throughout the droughttreatment. Shoot biomass was not different or slightly larger than thatof control. The ratio of water lost in 2 days over shoot dry weight onday 2 was lower for the myb68 mutant than control indicating trendstowards drought tolerance.

myb68 Mutant and 35S-gMYB68 and 35S-MYB68 Arabidopsis Transgenic LinesShow Salt Tolerance at Seedling Stage

Seeds were sterilized and placed on agar plates with ½ MS growth mediacontaining salt (200 mM NaCl) or no salt (optimal plates) with 6 platesper entry and 30 seeds per plate. After 3 days at 4 C plates were placedin the growth room at 22 C, and 18 hr lights (100 uE) for 16 days. After7 days plates were scored for germination and after additional 9 daysseedlings were scored for bleaching (% white seedling indicative ofstress). No differences were found between controls and transgenic linesor the mutant in germination on optimal plates and on salt plates. Butafter 16 days of salt exposure seedlings showed signs of stress bybecoming bleached. Results indicated that the myb68 mutant and thetransgenic 35S-Myb68 expressing lines had fewer bleached seedlings whichare indicative of lower sensitivity to salt stress.

Constitutive Expression of MYB68 in Arabidopsis Results in YieldProtection Following Heat Stress.

The 35S-Myb68 Arabidopsis plants were grown in 3″ pots with 3 plants perpot and 10 replicates per optimal treatment and 12 replicates per heattreatment. All plants were grown under optimal conditions until 2 daysinto flowering. At that point optimal plants remained in optimalconditions (22 C, 18 hr light of 200 uE, 70% RH) and the test group hada daily heat treatment applied by increasing temperature from 22° C. to45° C. over a 1 hr ramp period and maintaining that temperature for 1.5to 2.5 hr for five consecutive days. Plants recovered for a two dayperiod without applied stress after which stress was applied again foran additional three days (total of eight days of heat treatment).Following the heat treatments plants were maintained in optimalconditions till maturity together with the optimal group and final seedyield of both groups was determined. The results indicate that four35S-Myb68 transgenic lines (22-7, 20-11, 35-1 and 8-6) showed yieldprotection following the heat stress treatments that ranged from 8 to21% relative to controls.

Example 8: Functional Confirmation of Arabidopsis MYB-Subgroup14Sequences and Homologues from Other Species Produce the DesiredPhenotypes Such as Heat Tolerance Constitutive Expression of BnMYB68 inArabidopsis Results in Reduced Flower Abortion Following Heat Stress.

Two closely related Myb68 sequences were identified from Brassica. Theirexpression patterns differ in that one is expressed predominately in theroots (SEQ ID NO:54), the other in flower buds (SEQ ID NO:56). Plantshaving constructs expressing either of the BnMyb68 were produced andevaluated. Plants were grown in 2.25″ pots (1/pot) under optimalconditions (22 C, 50% RH, 17 hr light of 200 uE) until 3 days from firstopen flower. Plants were transferred from 22 C to 43 C for 2-2.5 hr (seetables below). Following this heat stress plants were returned tooptimal conditions at 22 C for a week. One week following the heatstress number of aborted flowers was counted. Transgenic lines of35S-BnMyb68(root) construct showed fewer aborted pods than its control(null). Two transgenic lines of 35S-BnMyb68(bud) construct showedreduced flower abortion following heat stress than their control (nullline).

Constitutive Expression of Soybean MYB84 or Arabidopsis MYB84 orArabidopsis MYB36 in Arabidopsis Results in Reduced Flower AbortionFollowing Heat Stress.

Over-expression constructs of soybean-Myb84 (GmMyb84) or ArabidopsisMyb84 (AtMyb84) or Arabidopsis Myb36 (AtMyb36) were made and transformedinto Arabidopsis plants functionally confirm the Myb-subgroup14homologues resulted in heat tolerance as demonstrated by reduced flowerabortion under heat stress. Transgenic plants were produced and the T2generation was used for an initial screen of heat tolerance.Subsequently, T3 homozygous transgenic plants are used for detailedphysiological assessment and confirmation of initial results.

The T2 seeds were plated on 0.5×MS agar plates with vitamins (1plate/flat). Each test group included a positive control, the originalheat tolerant mutant myb68, and a corresponding wild type. Seedlingswere transplanted to soil on day ten post germination. At the earlyflowering stage, plants were placed into a heating chamber at 45° C.,65% humidity) for 24-30 minutes. The plants were then placed back intothe growing chamber under normal growth conditions (17 h light/7 h dark,200 μE, 22° C., 70% humidity). Plants were examined on day thirty-twoand scored for aborted siliques, partially aborted siliques, deadmeristems or normal siliques. A heat stress was deemed effective if amajority of wild type plants had significant flower abortion Transgeniclines were assessed for gene expression by RT-PCR and demonstrated tohave elevated expression levels.

Constructs and transgenic plants are produced using homologous ofMyb-subgroup14 sequences from desired crop species, for example, rice,corn, wheat, soybean and cotton and evaluated for heat tolerance.

TABLE 13 flower abortion as % Construct of control 35S-Bn MYB68-bud 8435S-Bn-MYB68-root 82 35S-GmMYB84 58 35S-AtMYB84 77 35S-AtMYB36 81

This example demonstrates that Arabidopsis can be used as a model systemto assess and provide conformation that a Mub-subgroup14 sequence canprovide heat tolerance and that sequences identified from other plantspecies are functional in Arabidopsis.

Characterization of Arabidopsis Lines Expressing a 35S-AtMYB36 Construct

Fifteen homozygous transgenic lines at T3 were evaluated in a flowerabortion experiment. Plants (fourteen replicates per entry) were grownunder optimal conditions (18 hr light, 200 uE, 22 C, 60% RH) in 2.25″pots until four days from first open flower. At that point plants wereexposed to 1 hr treatment at 43 C. Following the heat treatment plantswere returned to optimal conditions for 7 more days. The impact of heattreatment on the pod formation was assessed at that point by countingthe aborted and damaged (short) pods in the region that was exposed tothe heat stress. Nine transgenic lines showed reduction in the totalnumber of damaged pods as compared to controls (wild type-Columbia andthe null-n77-4) (see table below). Three of the lines examined here alsoshowed reduced flower abortion at the T2 stage of screening. The myb68mutant, included as a positive control also showed a reduction in thetotal number of damaged pods as compared to its segregating null control(myb68-null).

Constitutive or Inducible Expression in Brassica napus of an ArabidopsisMYB68 Shows Reduced Flower Abortion Following Heat Stress

Five transgenic lines having an Arabidopsis Myb68 gene sequence underthe control of a constitutive promoter or a heat inducible promoter wereevaluated for heat stress tolerance under growth chamber conditions.These lines were at the T2 stage and were heterozygous, with theexception of the 01-105G-1-E line. Analysis of heterozygous linestypically produces greater variation than the same analysis performed onhomozygous lines. However, early analysis allows for screening andsubsequent analysis of the derived homozygous lines. Segregating nullsand the parent DH12075 were included as controls. The experiment wasarranged in a split-plot design with temperature as main factor andtransgenic line as subfactor. The plants were grown in 15 cm plasticpots filled with “Sunshine Mix #3” under approximately 500 μmol m⁻² s⁻¹photosynthetically active radiation at the top of the crop canopy.Molecular analysis was performed to confirm transgene presence. Twogroups of plants were included in the test; one group was grown underoptimum conditions (22/18° C. day/night temperature, 16-h photoperiod)throughout the growing period, while the second group was subjected toheat stress at 31° C. for 5 hr. per day (ramped-up from 18 to 31° C.,then back down to 18° C.). Heat stress conditions were initiated on thethird day following the first flower opening and for seven daysthereafter. The heat-stressed plants were then returned to optimumconditions until maturity. All racemes on the stressed plants weremarked at the beginning and end of the heat stress period, to indicatewhich flowers had been subjected to heat stress. Viable and aborted podson the marked racemes were counted and the pod abortion rate calculatedas the ratio of aborted pods to aborted plus viable pods underheat-stress conditions, expressed as percent. Seed yield was determinedafter harvest.

There were significant differences in flower abortion among differentlines, with two 35S-Myb68 lines showing significantly reduced abortionrate under heat stress compared to the parental line. Line 02-104G-4-Ahad a significantly lower abortion rate (33%) than its segregating null(48%) and DH12075 (61%). The abortion rate for line 02-104G-3-K (47%)was also significantly lower than that of the segregating null (66%) andDH12075. The homozygous line (01-105G-1-E) had a slightly lower abortionrate (53%) than its segregating null (58%) and the parental control.Within a population of transgenic lines a gradation of expression levelsand physiological response will exist. In part, the gradation ofvariation is a result of the site of integration of the gene constructand the local environment for gene expression at that locus. Thisgradation is expected and therefore, lines must be screened andevaluated in order to select the best performing lines. This process isone of routine to one skilled in the art.

Seed yield differed among the tested lines. The yield of threetransgenic lines, 02-104G-3-K, 02-104G-4-A and 01-105G-1-E, was similarto that of the parental line, however compared to it's own null thethree lines showed between 10% and 22% increase in seed yield. Twotransgenic lines (02-33H-1-V and 01-105G-3-G) appeared to under performcompared to the DH12075 parent however, compared to appropriate nulls,one line was significantly lower. Due to the zygosity of these linessuch variability is not unexpected.

A similar trend was found for seed number per raceme under heat stressconditions. Lines 02-104G-3-K, 02-104G-4-A and 01-105G-1-E had aslightly higher seed number per raceme but the other transgenic lines(02-33H-1-V and 01-105G-3-G) had significantly lower seed number perraceme compared the parent line. There were no significant differencesin 100 seed weight among the tested lines.

In general, two transgenic lines expressing the Arabidopsis Myb68 genedemonstrated significant protection against flower abortion during heatstress imposed at flowering. Additionally, three transgenic linesindicated a trend of increased seed yield.

TABLE 15 abortion Ab % Yield Construct Line rate % S.E. null Yield S.E.% Null 35S-MYB68 02-104G-3-K 47 4.8 71 0.71 0.188 122 02-104G-3-G null66 3.7 0.58 0.091 02-104G-4-A 33 7.6 69 0.75 0.153 110 02-104G-4-F null48 2.8 0.68 0.090 02-33H-1-V 79 1.9 116 0.31 0.159 44 02-33H-1-F null 684.3 0.71 0.090 P18.2-MYB68 01-105G-1-E 53 4.1 91 0.73 0.097 11601-105G-1-B null 58 3.0 0.63 0.090 01-105G-3-G 75 4.9 94 0.31 0.099 9601-105G-3-J null 80 3.6 0.32 0.097 Control DH12075 parent 61 2.4 0.620.106

Example 9: Identification of MYB-Subgroup14 Sequences and Homologues

Methods for identification of Arabidopsis MYB sequences, classificationof MYB sequences into designated subgroups and identification ofMYB-subgroup14 sequences are further described in Stracke et al., 2001and Kranz et al., 1998. MYB-subgroup14 sequences have been is defined asa nucleotide or protein sequence comprising an Arabidopsis thecharacteristics described herein. The MYB-subgroup14 is a R2R3 MYBsequence that additionally comprises a conserved motif or motifs asdescribed by the following patterns.

The general pattern (SEQ ID NO:266) provides a sequence that can be usedto identify a candidate MYB-subgroup14 sequence. At some positionsmultiple amino acid residues are permitted at a given location. Wheremultiple amino acids are permitted, the optional residues are indicatedwithin square brackets. Where a R2R3 MYB protein sequence fits thegeneral pattern it is likely to be a MYB-subgroup14 sequence. AMYB-subgroup14 sequence may be less than identical to the generalpattern, for example it may be 90%, 95% or 99% identical.

If a candidate MYB-subgroup14 sequence, matching the general patternfurther includes a match to the exclusive pattern (SEQ ID NO:267) thenthe sequence is a strong candidate for inclusion as a MYB-subgroup14sequence. The exclusive sequence (SEQ ID NO:267) defines the pattern ofamino acids that are present in MYB-subgroup14 sequences but may differin other R2R3 MYB proteins. Presence of the exclusive pattern within aMYB protein is a strong indicator that the MYB is a member of theMYB-subgroup14 family.

If a candidate MYB-subgroup14 sequence, matching the general patternfurther includes a match to the absolute pattern (SEQ ID NO:268) thenthe sequence is a strong candidate for inclusion as a MYB-subgroup14sequence. The Absolute pattern (SEQ ID NO:268) represents sequenceresidues present in all MYB-subgroup14 sequences analyzed to date.

For the general, exclusive, and absolute patterns (SEQ ID NOs:266-268)listed below, “X” denotes any amino acid, “X(N)”, where N is any number,denotes a string of the indicated number of “X”s, (i.e., X(23) denotes astring of 23 “X”s), where X is any amino acid. At some positionsmultiple amino acid residues are permitted at a given location. Wheremultiple amino acids are permitted, the optional residues are indicatedwithin square brackets.

General Pattern (SEQ ID NO: 266)M-G-R-X-P-C-C-D-[KR]-X X-[MV]-K-[RK]-G-X W-[SA]-X-[DQE]-E-D-X-X-[IL]-[RK]-X-[FY]-X-X-X-X-G-X-X-X-[SN]-W-I-X-X-P-X-[RK]-X-G-[IL]-X-R-C-G-[KR]-S-C-R-L-R-W-[IL]-N-Y-L-R-P-X-[IL]-[RK]-H-G-X-[FY]-[ST]-X-X-E-[DE]-X-X-[IV]-X-X-X-[FY]-X-X-X-G-S-[KR]-W-S-X-[MI]-A-X-X-[ML]-X-X-R-T-D-N-D-[ILV]-K-N-[HY]-W-[DN]-[ST]-[RK]-L-[RK]-[RK]-[RK] Exclusive Pattern (SEQ ID NO: 267)[RK]-X(9)G-X-X-I-X(28)-H-X(14)-[YF]-X(4)-S-X(23)- [RK] Absolute Pattern(SEQ ID NO: 268) G-X-W-X-X-X-E-D-X-X-[IL]-[RK]-X-X-X-X-X-X-G-X(23)-R-W-[IL]-N-Y-L-R-P-X-[IL]-[RK]-H-G-X-[FY]-X-X-X-E-[DE]-X(13)-W-X-X-X-A-X-X-X-X-X-R-T

MYB-subgroup14 sequences can be defined by the consensus sequence of SEQID NO:266. Positions have been identified in which the amino acidresidues are found exclusively or predominately in the MYB-subgroup14sequences. In the following description all position numbers are inreference to Arabidopsis MYB68 protein (SEQ ID NO:2) which correlates tothe general consensus sequence above, SEQ ID NO:266.

Within the R2 domain at position 26, a positively charged (K or R)residue is conserved in MYB-subgroup14. Although this amino acid is notexclusive to this subgroup at this position, the tendency for the restof the Arabidopsis MYB proteins is for a hydrophobic residue, with over50% of all MYB proteins having a hydrophobic isoleucine or valineresidue (Stracke et al., 2001).

At position 36, all members of subgroup 14 contain an insertion,resulting in an extra amino acid residue. This appears to be exclusiveto MYB-subgroup14. Glycine (G), a small hydrophobic residue, is theextra residue in all cases except in MYB87 (SEQ ID NO:32), whichcontains an asparagine residue at this position and a rice homologue(SEQ ID NO:194) which contains an argentine at this position.

At position 39, members of the MYB-subgroup14 predominantly contain ahydrophobic isoleucine residue. One exception to this is a ricehomologue identified as SEQ ID NO:191, which possesses a glutamine atthis position. Although other hydrophobic residues are generally foundin MYB proteins at this position, isoleucine appears to be exclusive toMYB-subgroup14. Additionally, positively charged residues (39% R), andpolar residues (23% N) are most prevalent at this position.

Within the R3 domain at position 68, all MYB-subgroup14 members containthe positively charged histidine residue. A positively charged residueappears at this position in all MYB proteins; however in contrast to theMYB-subgroup14, in other MYB proteins 73% contain an arginine (R) and17% contain a lysine residue.

At position 83, most members of MYB-subgroup14 contain an aromatichydrophobic residue (tyrosine Y, or phenylalanine F). The appearance ofan aromatic hydrophobic residue at this position seems to be exclusiveto MYB-subgroup14. The majority of MYB proteins have a histidine residue(87%). This histidine has been suggested to be a crucial residue in thehydrophobic core of the helix (Ogata et al., 1992). Through NMRanalysis, it appears to be in contact with two of the criticaltryptophan residues. The absence of a histidine at position 83 does notnecessarily exclude it as a member of MYB-subgroup14 as members havebeen identified that do possess a histidine residue, for example SEQ IDNO:169 and SEQ ID NO:225.

At position 88, members of MYB-subgroup14 contain the polar serineresidue, while almost all other MYB proteins (91%) contain the polarasparagine residue. A serine residue appears to be exclusive to thissubgroup. The absence of a serine at position 88 does not necessarilyexclude it as a member of MYB-subgroup14 as at least one MYB-subgroup14member has been identified that possesses a phenylalanine residue inposition 88, for example SEQ ID NO:256.

At position 112, members of MYB-subgroup14 contain a positively chargedarginine or lysine residue. The majority of MYB proteins also containpositively charged residues at this position, however, histidine (48%)is the most prevalent residue found. The absence of a arginine atposition 112 does not necessarily exclude it as a member ofMYB-subgroup14 as at least one MYB-subgroup14 member has been identifiedthat possesses a glutamic acid residue in position 112, for example SEQID NO:68 contains a glutamic acid residue and SEQ ID NO:191 contains athreonine.

Variation within a R2R3 domain is permissible as shown in the identifiedconsensus sequences. A R2R3 domain of a MYB-subgroup14 sequence may be90% homologous, preferably 95% homologous or more preferably 99%homologous to the consensus sequence presented.

The Addition of S1 and S2 Motifs

Within MYB-subgroup14 the MYB68 and MYB84 sequences contain two furtherconserved motifs identified as S1 (SFSQLLLDPN) (SEQ ID NO:269) and S2(TSTSADQSTISWEDI) (SEQ ID NO:270). These motifs are found in bothArabidopsis and Brassica MYB68, MYB36 and MYB84 sequences and show atleast 70% homology within the amino acid sequence. Additionally,homology to the S1 or S2 motifs was found to exist in orthologs inBrassica napus (Canola), Brassica rapa (Cabbage), Brassica oleracea,Raphanus raphanistrum (Radish), and homology to the S2 region inPoncirus trifoliate (Orange), and weak homology in a Medicago trunculatahomologue and Vitis Vinifera (Grape) homologue within the S2 region.

For inclusion as a S1 or S2 motif target sequences show homology of atleast 70%, more preferably 80% and most preferably 95%.

The MYB68 and MYB84 sequences from species other than Arabidopsis andBrassica may not contain a S1 and S2 motif but may still be classifiedas a MYB subgroup-14 sequence based on sequence analysis and inclusionof other criteria.

Identification of MYB-Subgroup14 Members, Including MYB68, Homologues

Homologues of an Arabidopsis MYB-subgroup14 sequence (Table 1) or adesired MYB68 (SEQ ID NO:1, SEQ ID NO:2), can be found using a varietyof public or commercial software that is known to those skilled in theart. Blast alignments can be performed and putative sequencesidentified. Searches can be performed as outlined herein. The tophomologues are determined using programs such as tblastn, tblastp,searches against available databases in NCBI, such as the EST, GSS, HTGand chromosomal databases, as well as other genomic databases, such asthe TIGR unigene database, Cucurbit genomics database, Sunflower andLettuce, Medicago truncatula (International Medicago Genome AnnotationGroup), SGN, and Orange. In instances where species were more highlydivergent, the alignment parameters such as Gap costs, matrix values canbe appropriately changed.

To confirm the top hit to AtMYB68 in each species is in fact a MYB68homologue, a reciprocal blast can be performed, in which the homologueis blasted against all Arabidopsis proteins. In many cases thehomologue's closest Arabidopsis hit is to one of MYB68's gene familymembers (a MYB subgroup-14), instead of MYB68 itself. In cases where thehomologue is closest to an Arabidopsis protein outside of the MYB68 genefamily, the homologue is assessed not to be a MYB-subgroup14 member.

Open reading frames may be determined using programs such as “getorf”from the EMBOSS program, or ESTScan.

Methods for identification of MYB sequences, classification of MYBsequences into designated subgroups and identification of MYB-subgroup14sequences are further described in Stracke et al., 2001 and Kranz etal., 1998.

Weakly conserved homologues between highly divergent species are oftennot found using traditional blast methods. In such cases, conservedmotifs, domains and fingerprints will exist between homologues, and aregood predictors of functional homology. Many programs exist that areproficient at finding conserved domains across species using hiddenmarkov models, position-specific-scoring matrices, and patterns.PSI-Blast, PRATT, PHI-Blast, and HMMBuild/HMMSearch.

PRATT is a tool provided by the PROSITE database. It generates conservedpatterns from a group of conserved proteins. PRATT was used to determinea conserved pattern between MYB68 and its closest homologues.ScanProsite and PHI-BLAST was then used to look for the conservedpattern in the Swiss-Prot and NCBI protein databases respectively. Thesearch results are limited to alignments that also contain the pattern.

HMMBuild was used to build a hidden markov model using the MYB68 and itshomologues. HMMSearch was used to scan NCBI's protein database using thehidden markov model. Similar proteins to the basic blastp search werefound.

Utilizing the above methods, MYB68 homologues were found in over 50different plant species. Homology was restricted in most cases to theN-terminal MYB DNA binding domain. Homology in the less conservedC-terminal region existed in genes found in Brassica napus (Canola),Brassica rapa (Cabbage), Brassica oleracea, Raphanus raphanistrum(Radish), Poncirus trifoliate (Orange), and weak homology in genes foundin Medicago trunculata homologue and Vitis Vinifera (Grape) within theS2 region.

The Poncirus trifoliate and Brassica rapa genes were identified bydownloading strong EST hits, and assembling them using CAP3. Theresulting contigs did not code for a complete protein. The contigsequence from Orange coded for a partial protein spanning only the S2region, and in Brassica rapa, a partial 202 aa protein spanning from1-202 in AtMYB68 was found.

A variety of programs have characterized the MYB domain with profiles,patterns, and hidden markov models. To confirm the presence of the MYBdomain in a unknown sequence, a sequence can be searched against theseprofiles. InterProScan is particularly useful as it provides aninterface to query 13 programs simultaneously. The databasesincorporated in InterPro include

a) ProDom: a database of protein domain families. Built by clusteringhomologous segments from Swiss-Prot/Trembl database, followed byrecursive PSI-BLAST searches.b) HMMTIGR: Protein families represented by Hidden Markov Models.c) TMHMM: Prediction of transmembrane helices in proteins.d) FPrintScan: Searches the PRINTS database of fingerprints.Fingerprints are protein families represented by multiple motifs.e) ProfileScan: Profiles from family related sequences.f) HMMPanther: A database of hidden markov modelsg) HMMPIR: Hidden markov models based on evolutionary relationship ofwhole proteins.h) ScanRegExp: Scans the prosite database of patterns and profilesi) Gene3D: a database of proteins containing functional informationj) HMMPfam: Protein domain families represented by hidden markov models.k) Superfamily: a database of structural and functional proteinannotations for all completely sequenced organisms.l) HMMSmart: Allows the identification of mobile domains based on hiddenmarkov models.m) SignalIP: predicts the presence and location of signal peptidecleavage sites in amino acid sequences.

Blocks are multiply aligned ungapped segments corresponding to the mosthighly conserved regions of proteins. The MYB domain is represented bythree blocks. As expected AtMYB68 contained each of these three blocks,as well as 1 of the 5 Wos2 blocks.

The invention is based in part on the discovery of plants that are heatstress tolerant. The gene responsible for the heat tolerant phenotypehas been determined and shown to be a MYB68 gene. Methods of producing aheat tolerant transgenic plant are disclosed herein. Specifically theinvention identifies a transcription factor gene family, specificallythe MYB gene family, and in particular a MYB-subgroup14 that whenexpressed in plants results in plants that are heat stress tolerant andhave an increased yield following a heat stress or display tolerance todrought stress or salt stress.

Example 10: Identification of MYB68 Homologues

Homologues from the same plant, different plant species or otherorganisms were identified using database sequence search tools, such asthe Basic Local Alignment Search Tool (BLAST) (Altschul et al. (1990) J.Mol. Biol. 215:403-410; and Altschul et al. (1997) Nucl. Acid Res. 25:3389-3402). The tblastn or blastn sequence analysis programs wereemployed using the BLOSUM-62 scoring matrix (Henikoff, S. and Henikoff,J. G. (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919). The output ofa BLAST report provides a score that takes into account the alignment ofsimilar or identical residues and any gaps needed in order to align thesequences. The scoring matrix assigns a score for aligning any possiblepair of sequences. The P values reflect how many times one expects tosee a score occur by chance. Higher scores are preferred and a lowthreshold P value threshold is preferred. These are the sequenceidentity criteria. The tblastn sequence analysis program was used toquery a polypeptide sequence against six-way translations of sequencesin a nucleotide database. Hits with a P value less than −25, preferablyless than −70, and more preferably less than −100, were identified ashomologous sequences (exemplary selected sequence criteria). The blastnsequence analysis program was used to query a nucleotide sequenceagainst a nucleotide sequence database. In this case too, higher scoreswere preferred and a preferred threshold P value was less than −13,preferably less than −50, and more preferably less than −100.

Alternatively, a fragment of a sequence from SEQ ID NO: 1, 3, 5, 7, 9,11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 38, 40, 42, 44,46, 48, 50, 52, 54, 56, 58, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79,81, 83, 85, 87, 88, 90, 91, 92, 94, 96, 98, 100, 102, 104, 106, 108,109, 110, 112, 114, 116, 118, 120, 122, 124, 125, 127, 129, 130, 132,134, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 156, 158,160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186,188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 211, 213,214, 216, 217, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238,240, 242, 244, 246, 247, 249, 251, 253, 255, 257, 259, 261, 263 and 265is ³²P-radiolabeled by random priming (Sambrook et al., (1989) MolecularCloning. A Laboratory Manual, 2^(nd) Ed., Cold Spring Harbor LaboratoryPress, New York) and used to screen a plant genomic library (theexemplary test polynucleotides) As an example, total plant DNA fromArabidopsis thaliana, Nicotiana tabacum, Lycopersicon pimpinellifolium,Prunus avium, Prunus cerasus, Cucumis sativus, or Oryza sativa areisolated according to Stockinger al (Stockinger, E. J., et al., (1996),J. Heredity, 87:214-218). Approximately 2 to 10 μg of each DNA sampleare restriction digested, transferred to nylon membrane (MicronSeparations, Westboro, Mass.) and hybridized. Hybridization conditionsare: 42.degree. C. in 50% formamide, 5×SSC, 20 mM phosphate buffer1×Denhardt's, 10% dextran sulfate, and 100 μg/ml herring sperm DNA. Fourlow stringency washes at RT in 2×SSC, 0.05% sodium sarcosyl and 0.02%sodium pyrophosphate are performed prior to high stringency washes at55° C. in 0.2.times.SSC, 0.05% sodium sarcosyl and 0.01% sodiumpyrophosphate. High stringency washes are performed until no counts aredetected in the washout according to Walling et al. (Walling, L. L., etal., (1988) Nucl. Acids Res. 16:10477-10492).

Example 11: Identification of MYB68 Technical Features

A MYB68 gene can be identified by identifying genes that have highhomology to an Arabidopsis MYB68 (SEQ ID NO:1). In addition to havinghomology of the nucleotide or amino acid sequence, one may identifycandidate genes that share conformational protein structure. Suchstructural motifs may assist in identification of related proteins andtheir structure and function relationships.

Example 12: Functional Confirmation of Homologues

Candidate homologues are introduced into Arabidopsis and assessed forheat tolerance. Genes disclosed as SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15,17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 38, 40, 42, 44, 46, 48, 50,52, 54, 56, 58, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85,87, 88, 90, 91, 92, 94, 96, 98, 100, 102, 104, 106, 108, 109, 110, 112,114, 116, 118, 120, 122, 124, 125, 127, 129, 130, 132, 134, 135, 137,139, 141, 143, 145, 147, 149, 151, 153, 155, 156, 158, 160, 162, 164,166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192,194, 196, 198, 200, 202, 204, 206, 208, 210, 211, 213, 214, 216, 217,218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244,246, 247, 249, 251, 253, 255, 257, 259, 261, 263 and 265 are expressedin Arabidopsis plants and heat tolerance assessed as described herein.Optionally, the expression of If a candidate MYB-subgroup14 sequence,matching the general pattern further includes a match to the exclusivepattern then the sequence is a strong candidate for inclusion as aMYB-subgroup14 genes or MYB68 genes can be evaluated in anytransformable species, for example, Brassica, maize, cotton, soybean orrice. Examples of such functional testing have been provided in thisdisclosure.

It is noted that some of the sequences disclosed herein are not fulllength. Full length sequences can be obtained by standard molecularmethods known to those of skill in the art. The full length sequencescan then be expressed as described herein.

What is claimed is:
 1. A method of producing a heat stress tolerantplant, comprising: a) providing a nucleic acid encoding a MYBsubgroup-14 polypeptide selected from the group consisting of MYB36,MYB37, MYB38, MYB68, MYB84, and MYB87; b) inserting said nucleic acidinto a vector; c) transforming a plant, a tissue culture, or a plantcell with said vector to obtain a transformed plant, tissue culture, orplant cell with an increased expression of said MYB subgroup-14polypeptide; d) growing said transformed plant or regenerating a plantfrom said transformed tissue culture or plant cell, wherein a heatstress tolerant plant is produced which has an increased heat stresstolerance as compared to a wild type plant.
 2. The method according toclaim 1, wherein said MYB subgroup-14 polypeptide is selected from thegroup consisting of MYB36, MYB68, and MYB84.
 3. The method according toclaim 1, wherein said MYB subgroup-14 polypeptide is MYB68.
 4. Themethod according to claim 1, wherein said MYB subgroup-14 polypeptide isMYB36.
 5. The method according to claim 1, wherein said MYB subgroup-14polypeptide is MYB37.
 6. The method according to claim 1, wherein saidvector comprises a constitutive promoter or an inducible promoter.
 7. Aheat stress tolerant transgenic plant produced by the method of claim 1,wherein said transgenic plant contains a vector or an expressioncassette comprising said nucleic acid encoding said MYB subgroup-14polypeptide resulting in increased expression of said MYB subgroup-14polypeptide, and has an increased tolerance to heat stress, and whereinsaid MYB subgroup-14 polypeptide is MYB68.
 8. The transgenic plant ofclaim 7, wherein said transgenic plant has an increased seed yieldrelative to a wild type control.
 9. A transgenic seed produced by thetransgenic plant of claim 7, wherein said transgenic seed is transformedwith said nucleic acid encoding MYB68 and produces a heat stresstolerant plant which expresses said nucleic acid encoding said MYB68 andhas an increased heat stress tolerance.
 10. A heat stress toleranttransgenic plant produced by the method of claim 1, wherein saidtransgenic plant contains a vector or an expression cassette comprisingsaid nucleic acid encoding said MYB subgroup 14 polypeptide resulting inincreased expression of said MYB subgroup-14 polypeptide, and has anincreased tolerance to heat stress, and wherein said MYB subgroup 14polypeptide is MYB36.
 11. The transgenic plant of claim 10, wherein saidtransgenic plant has an increased seed yield relative to a wild typecontrol.
 12. A transgenic seed produced by the transgenic plant of claim10, wherein said transgenic seed is transformed with said nucleic acidencoding MYB36 and produces a heat stress tolerant plant which expressessaid nucleic acid encoding MYB36 and has an increased heat stresstolerance.